Cyclic compound, method for producing cyclic compound, and method for modifying biological molecule

ABSTRACT

The invention aims in establishing a method for modifying biomolecules using a reaction that efficiently modifies biomolecules and is widely applicable. The invention thus provides a cyclic compound containing two triazole rings formed by adding and ligating an azide compound possessing an azido group to each of the two carbon-carbon triple bond sites of an eight-membered cyclic skeleton of a cyclic diyne compound by a double click reaction; a method for producing a cyclic compound using a double click reaction; and a method for modifying biomolecules.

TECHNICAL FIELD

The present invention relates to a cyclic compound, a method forproducing the cyclic compound by a strain-promoted double-clickreaction, and a method for modifying biomolecules.

BACKGROUND ART

Chemical modification of biomolecules is performed to clarify themechanism for the activity expression of biologically active substancesor the function of biomolecules. There is known a method for selectivelymodifying glycoconjugates with fluorescent dyes, etc., which involvesintroducing an azido group into biomolecules, e.g., glycoconjugates atthe cell membrane surface, etc. and reacting the azido group withtriarylphosphines having a fluorescent functional group, etc. as a probeto ligate them through covalent bond (e.g., see Non-Patent Documents 1to 3).

Also, a method using a single click reaction in which a single azidecompound is added and ligated to an alkyne is also known. In this case,probe-bearing biomolecules can be identified by reacting the azido groupintroduced into the target biomolecule with an alkyne compound as aprobe. As examples of the click reaction, a reaction using Cu(I)catalyst and a catalyst-free reaction are both known (e.g., seeNon-Patent Documents 4 to 13).

CITATION LIST Non Patent Literature

-   [Non-Patent Document 1] E. Saxon, C. R. Bertozzi, Science, 2000,    287, 2007-2010-   [Non-Patent Document 2] K. L. Kiick, E. Saxon, D. A. Tirrell, C. R.    Bertozzi, Proc. Natl. Acad. Sci. USA, 2002, 99, 19-24-   [Non-Patent Document 3] J. A. Prescher, D. H. Dube, C. R. Bertozzi,    Nature, 2004, 430, 873-877; Nicholas J. Agard, Jennifer A. Prescher,    and Carolyn R Bertozzi, J. Am. Chem. Soc. 126, 15046-15047, 2004-   [Non-Patent Document 4] N. J. Agard, J. A. Prescher, C. R.    Bertozzi, J. Am. Chem. Soc. 2004, 126, 15046-15047-   [Non-Patent Document 5] N. J. Agard, J. M. Baskin, J. A.    Prescher, A. Lo, C. R. Bertozzi, ACS Chem. Biol. 2006, 1, 644-648-   [Non-Patent Document 6] J. M. Baskin, J. A. Prescher, S. T.    Laughlin, N. J. Agard, P. V. Chang, I. A. Miller, A. Lo, J. A.    Codelli, C. R. Bertozzi, Proc. Natl. Acad. Sci. USA 2007, 104,    16793-16797-   [Non-Patent Document 7] J. A. Codelli, J. M. Baskin, N. J.    Agard, C. R. Bertozzi, J. Am. Chem. Soc. 2008, 130, 11486-11493-   [Non-Patent Document 8] S. T. Laughlin, J. M. Baskin, S. L.    Amacher, C. R. Bertozzi, Science 2008, 320, 664-667-   [Non-Patent Document 9] X. Ning, J. Guo, M. A. Wolfert, G J. Boons,    Angew. Chem. 2008, 120, 2285-2287; Angew. Chem. Int. Ed. 2008, 47,    2253-2255-   [Non-Patent Document 10] A. A. Poloukhtine, N. E. Mbua, M. A.    Wolfert, G-J. Boons, V. V. Popik, J. Am. Chem. Soc. 2009, 131,    15769-15776-   [Non-Patent Document 11] S. T. Laughlin, C. R. Bertozzi, ACS Chem.    Biol. 2009, 4, 1068-1072-   [Non-Patent Document 12] M. F. Debets, S. S. van Berkel, S.    Schoffelen, F. P. J. T. Rutjes, J. C. M. van Hest, F. L. van Delft,    Chem. Commun. 2010, 46, 97-99-   [Non-Patent Document 13] P. V. Chang, J. A. Prescher, E. M.    Sletten, J. M. Baskin, I. A. Miller, N. J. Agard, A. Lo, C. R.    Bertozzi, Proc. Natl. Acad. Sci. USA 2010, 107, 1821-1826

SUMMARY OF THE INVENTION Technical Problem

The modification reaction between an azido group and a phosphinederivative described above requires a plurality of steps including atleast a probing reaction of the phosphine derivative and a ligationreaction of the azido group and the phosphine derivative. For thisreason, it is difficult to modify biomolecules efficiently. Inparticular, the reaction rate between the azido group and phosphinederivative is generally slow, and it is therefore more difficult toaccelerate the modification reaction. Furthermore, phosphine derivativesare susceptible to oxidation and hence, biomolecules cannot always bemodified with a high efficiency.

Also in the single click reaction of adding a single azide compound toan alkyne, previous probing of the alkyne molecule is required each timedepending upon the purpose of experimentation. It is therefore difficultto modify biomolecules in a small number of steps. In addition, thecatalyst-free single click reaction involves complicated substratesynthesis and when catalysts are used, the catalysts show cytotoxicity,and so on. Thus, the single click reaction is disadvantageous in thatthe reaction is not widely available to modification of biomolecules.

Under the circumstances above, there is a need for cyclic compoundsobtained by a reaction for efficiently modifying biomolecules using asubstrate readily available in a less number of steps, which reaction isbroadly applicable, a method for producing cyclic compounds using such areaction, and a method for modifying biomolecules.

Solution to Problem

The present inventors have found that by taking advantage of a doubleclick reaction using highly strained diynes, biomolecules can beefficiently modified in a less number of steps and this method isbroadly applicable. Based on the finding, the present invention has beenaccomplished. The present invention provides cyclic compounds, a methodfor producing cyclic compounds by the double click reaction, and amethod for modifying biomolecules.

<1> A cyclic compound comprising a cyclic skeleton and two triazolerings sharing carbon-carbon double bond sites with the cyclic skeleton.

<2> The cyclic compound according to <1> above, which contains thetriazole rings formed by adding and ligating an azide compound having anazido group to each of the two carbon-carbon triple bond sites on thecyclic skeleton in a cyclic diyne compound by a double click reaction.

<3> The cyclic compound according to <1> or <2> above, which containsthe cyclic skeleton of an 8-membered ring.

<4> The cyclic compound according to any one of <1> to <3> above, whichfurther contains a benzene ring and/or heteroaromatic ring sharing thecarbon-carbon double bond sites with the cyclic skeleton.

<5> The cyclic compound according to any one of <1> to <4> above, whichis represented by formula (1) or (2) below:

(in formula (1), each R independently represents hydrogen or ahydrocarbon group, and each n independently represents an integer of 0to 4, preferably an integer of 0 or 1), and,

(in formula (2), each R independently represents hydrogen or ahydrocarbon group, and each n independently represents an integer of 0to 4, preferably an integer of 0 or 1).

<6> A method for producing a cyclic compound, which comprises adding andligating an azide compound having an azido group to each of the twocarbon-carbon triple bond sites in a cyclic diyne compound by a doubleclick reaction to produce a cyclic compound containing two triazolerings.

<7> A method for modifying a biomolecule, which comprises adding andligating the azido group of an azide compound as a probe and the azidogroup incorporated into the biomolecule to each of the two carbon-carbontriple bond sites in a cyclic diyne compound by a double click reactionto produce a cyclic compound.

<8> The modifying method according to <7> above, wherein the cyclicdiyne compound has an 8-membered cyclic skeleton containing twocarbon-carbon triple bond sites.

<9> The modifying method according to <8> above, wherein the cyclicdiyne compound further has a benzene ring and/or heteroaromatic ringsharing the carbon-carbon double bond sites with the cyclic skeleton.

<10> The modifying method according to any one of <7> to <9> above,wherein the cyclic diyne compound is represented by formula (3) below:

(in formula (3), each R independently represents hydrogen or ahydrocarbon group, and each n independently represents an integer of 0to 4, preferably an integer of 0 or 1).

<11> The modifying method according to any one of <7> to <10> above,wherein the addition reaction of the azide compound and the biomoleculeproceeds without using any catalyst.

<12> The modifying method according to any one of <7> to <11> above,wherein the double click reaction is performed in the co-presence of theazide compound and the biomolecule.

<13> The modifying method according to any one of <7> to <12> above,wherein the biomolecule is added to the cyclic diyne compound, theunreacted cyclic diyne compound is removed and the azide compound isadded to the biomolecule-added cyclic diyne compound only.

Advantageous Effects of Invention

According to the present invention, biomolecules can be efficientlymodified in a less number of steps by a double click reaction using ahighly strained diyne which can be readily synthesized. In addition, thedouble click reaction is applicable to a variety of compounds, and suchan efficient modifying method can be utilized broadly.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the calculated results of activation energies andtransition states in the SPDC reaction of highly strained diyne withmethyl azide.

FIG. 2 shows decay curves of the azide compound (benzyl azide) in theSPDC reaction shown by scheme (14).

FIG. 3 shows a straight line obtained by plotting the pseudo-first orderrate constants versus the azide compound with different concentrationsin the SPDC reaction of FIG. 2 (pseudo-first order rate constants in theSPDC reaction).

FIG. 4 shows the results of SDS-PAGE analysis of fluorescence-labeledHaloTag proteins by the SPDC reaction using a highly strained diyne andTESRA-PEO₃-azide.

FIG. 5 shows the results of mass spectrometry of fluorescence-labeledHaloTag proteins with TESRA-HaloTag ligand.

FIG. 6 shows the results of mass spectrometry of fluorescence-modifiedHaloTag proteins by the SPDC reaction using the highly strained diyneand TESRA-PEO₃-azide.

FIG. 7 shows the results of fluorescence labeling test ofglycoconjugates at the living cell surface by the SPDC reaction usingthe highly strained diyne and TESRA-PEO₃-azide (modification of cellsurface with the diyne and the azide).

FIG. 8 shows the results of fluorescence labeling test ofglycoconjugates on the surface of living cells by the SPDC reactionusing a highly strained diyne and Alexa Fluor 488 azide.

FIG. 9 shows the results of fluorescence labeling test ofglycoconjugates on the living cell surface by the SPDC reaction and thesingle click reaction, respectively.

FIG. 10 shows the results of cytotoxicity assay of the highly straineddiyne used in the SPDC reaction.

DESCRIPTION OF EMBODIMENTS 1. Cyclic Compound and Method for ProducingCyclic Compound by Double Click Reaction

The cyclic compound of the present invention comprises the cyclicskeleton and two 1,2,3-triazole ring sharing the carbon-carbon doublebond sites with the cyclic compound. The cyclic compound is produced viaa double click reaction by adding an azide compound with an azido groupto the cyclic diyne compound at each of the two carbon-carbon triplebond sites to form two 1,2,3-triazole rings which are fused rings.

1A. Double Click Reaction

The reaction of adding two azide compounds to one diyne compound so asto add the azide compounds to each of the two carbon-carbon triple bondsites of the cyclic diyne compound is called a double click reaction. Inthe double click reaction shown by general reaction scheme (I) below,different azide compounds can be added to the two carbon-carbon triplebond sites to ligate three molecules spontaneously.

An 8-membered cyclic diyne compound is used as a substrate for thedouble click reaction. This is because the 8-membered cyclic diynecompound is highly strained to promote the double click reaction. Assuch, the double click reaction in which distortion of the cyclic diynecompound promote is referred to as the SPDC reaction (Strain-PromotedDouble-Click Reaction).

In the double click reaction including the SPDC reaction, an excessamount of the azide compound is used based on the diyne compound. Forexample, 2 to 10 equivalents, preferably 2 to 5 equivalents, morepreferably 2 to 3 equivalents, of the azide compound is used based on 1equivalent of the diyne compound.

In the double click reaction, various types of solvents can be used.That is, mainly lower alcohols such as methanol, ethanol, etc., organicsolvents such as acetonitrile, tetrahydrofuran, dimethylsulfoxide,N,N-dimethylformamide, pyridine, dichloromethane, chloroform, benzene,toluene, diethyl ether, dioxan, acetone, ethyl acetate, hexane, etc.,water or a buffer aqueous solution of appropriate pH, a solvent mixturethereof, can be used, as long as the azide compounds and cyclic diynecompounds are suitably soluble therein.

The double click reaction is promoted by distortion of the cyclic diynecompound and proceeds under relatively mild conditions. Morespecifically, the double click reaction can be performed around roomtemperature in the absence of a catalyst, e.g., a copper catalyst usedto expedite the double click reaction. In this regard, however, heatingmay be performed, if necessary, by elevating to approximately 150° C. orby using a suitable solvent, the reaction may also be performed underlow temperature conditions of approximately −100° C. The reaction timefor the double click reaction may vary depending upon substrate and itsconcentration or reaction solvent, etc. but generally rangesapproximately from one minute to one day, normally approximately from 10minutes to 2 hours.

1B. Cyclic Diyne Compound ((A) in general scheme (I) described above)

Preferably, the cyclic diyne compound is strained and furthermore areasonable stability is required. Therefore, at least an 8-memberedcyclic diyne compound is more preferred than unstable cyclic diynecompounds having a 7-membered ring or less. In particular, the cyclicdiyne compound having an 8-membered cyclic skeleton is desired. Thecyclic diyne compound which can be used further includes a heterocycliccompound containing oxygen, nitrogen, sulfur, etc.

Cyclic triyne compounds, cyclic tetrayne compounds and cyclic compoundshaving a larger number of the carbon-carbon triple bonds can also beused for the double click reaction, triple click reaction, quadrupleclick reaction, and so on, as far as they can be present stably. In thecyclic skeleton having, e.g., a 9-membered ring, triyne and tetraynecompounds can be present stably.

In the cyclic diyne compound, it is preferred to have a cyclic skeletonhaving preferably an 8-membered ring containing two carbon-carbon triplebonds and a benzene ring, etc. sharing the carbon-carbon double bondsites with the cyclic skeleton described above. This is because thecyclic diyne compound can attain its planarity due to such a benzene toprovide a good reactivity with an azide which is a 1,3-dipolar compound.The cyclic diyne compound preferably contains two benzene rings. Inaddition to the benzene ring, the following cyclic diyne compounds canalso be used in the double click reaction (SPDC reaction); a benzenering which is substituted with a hydrocarbon group, etc.; aheteroaromatic ring including a pyridine ring, a pyrimidine ring, apyrazine ring, a furan ring, a thiophene ring, a pyrrole ring, animidazole ring, a triazole ring, an oxazole ring, a thiazole ring, etc.,which may be substituted; a polycyclic type diyne compound formed byfusing a plurality of these heteroaromatic rings including a naphthalenering, an anthracene ring, a quinoline ring, an isoquinoline ring, aquinoxaline ring, a benzofuran ring, a benzothiophene ring, an indolering, a benzimidazole ring, etc. The aromatic rings described above mayalso be combined and provided for use, for example, as a cyclic diynecompound having each one of these aromatic rings.

The substituent (R in general scheme (I) described above) contained inthe cyclic diyne compound includes a hydrocarbon group such as a C1-10alkyl group (methyl, ethyl, propyl, etc.), a C2-10 alkenyl group(ethenyl, 1-propenyl, 2-propenyl, etc.), a C2-10 alkynyl group (ethynyl,1-propynyl, 2-propynyl, etc.), a C3-10 cycloalkyl group (cyclopropyl,cyclobutyl, cyclopentyl, etc.), a C3-10 cycloalkenyl group(2-cyclopenten-1-yl, 3-cyclopenten-1-yl, 2-cyclohexen-1-yl, etc.), aC4-10 cycloalkadienyl group (2,4-cyclopentadien-1-yl,2,4-cyclohexadien-1-yl, 2,5-cyclohexadien-1-yl, etc.), a C6-14 arylgroup (phenyl, naphthyl, anthryl, etc.), a C7-13 aralkyl group (benzyl,phenethyl, naphthylmethyl, etc.), a C8-13 arylalkenyl group (styryl,etc.), a C3-10 cycloalkyl-C1-6 alkyl group (cyclohexylmethyl, etc.),etc.; hydroxy group, a C1-10 alkoxyl group, a C1-10 alkoxycarbonylgroup, thiol group, a C1-10 alkylthio group, amino group, a di- ormono-C1-10 alkylamino group, carboxyl group, amido group, thioamidogroup, thiol group, ether group, ester group, sulfo group, sulfonamidogroup and a halogen; a substituted or unsubstituted heteroaromatic ringsuch as a pyridine ring, a pyrimidine ring, a pyrazine ring, a furanring, a thiophene ring, a pyrrole ring, an imidazole ring, a triazolering, an oxazole ring, a thiazole ring, etc.; a polycyclic ring systemformed by a plurality of the heteroaromatic rings described above and aC6-14 aryl group, etc.; a substituent containing at least one ofhalogen, nitrogen, oxygen, sulfur, etc. in one of the substituentsabove; a substituent(s) further containing at least one of hydroxygroup, amino group, carboxyl group, amido group, thioamido group, thiolgroup, ether group, ester group, sulfo group, sulfonamido group, etc. inone of the substituents above; etc. Among them, preferred examples ofthe substituent R are a hydrocarbon group including a C1-10 alkyl group(methyl, ethyl, propyl, etc.), a C2-10 alkenyl group (ethenyl,1-propenyl, 2-propenyl, etc.), a C2-10 alkynyl group (ethynyl,1-propynyl, 2-propynyl, etc.), a C3-10 cycloalkyl group (cyclopropyl,cyclobutyl, cyclopentyl, etc.), a C3-10 cycloalkenyl group(2-cyclopenten-1-yl, 3-cyclopenten-1-yl, 2-cyclohexen-1-yl, etc.), aC4-10 cycloalkadienyl group (2,4-cyclopentadien-1-yl,2,4-cyclohexadien-1-yl, 2,5-cyclo hex adien-1-yl, etc.), a C6-14 arylgroup (phenyl, naphthyl, anthryl, etc.), a C7-13 aralkyl group (benzyl,phenethyl, naphthylmethyl, etc.), a C8-13 arylalkenyl group (styryl,etc.), a C3-10 cycloalkyl-C1-6 alkyl group (cyclohexylmethyl, etc.),etc. More preferred examples of the substituent R are a C1-10 alkylgroup (methyl, ethyl, propyl, etc.), a C2-10 alkenyl group (ethenyl,1-propenyl, 2-propenyl, etc.), a C2-10 alkynyl group (ethynyl,1-propynyl, 2-propynyl, etc.), a C3-10 cycloalkyl group (cyclopropyl,cyclobutyl, cyclopentyl, etc.), a C3-10 cycloalkenyl group(2-cyclopenten-1-yl, 3-cyclopenten-1-yl, 2-cyclohexen-1-yl, etc.) and aC4-10 cycloalkadienyl group (2,4-cyclopentadien-1-yl,2,4-cyclohexadien-1-yl, 2,5-cyclohexadien-1-yl, etc.). In thesesubstituents, the carbon number is generally preferably within the rangedescribed above to avoid steric hindrance that might prevent theaddition reaction of an azido molecule. The substituent(s) describedabove is/are bound to the cyclic diyne compound, either via a C1-10alkyl linker, etc. containing, e.g., a single or plurality of oxygenatoms, amido bonds or ester bonds, or directly.

Based on the foregoing, preferred examples of the cyclic diyne compoundas the substrate for the strain-promoted double click reaction (SPDCreaction) are sym-dibenzo-1,5-cyclooctadiene-3,7-diyne(5,6,11,12-tetrahydrodibenzo[a,e]cyclooctene),5,6,11,12-tetrahydro-1,4,7,10-tetramethyldibenzo[a,e]cyclooctene,5,6,11,12-tetrahydro-2,3,8,9-tetramethoxydibenzo[a,e]cyclooctene,1,7-dibutyl-5,6,11,12-tetrahydrodibenzo[a,e]cyclooctene,6,7,14,15-tetrahydrocycloocta[1,2-b:5,6-b′]dinaphthalene, etc., and morepreferably, the cyclic diyne compound is, e.g.,sym-dibenzo-1,5-cyclooctadiene-3,7-diyne(5,6,11,12-tetrahydrodibenzo[a,e]cyclooctene).

1C. Azide Compound ((B) in General Scheme (I) Above)

Almost all organic azide compounds are usable as the azide compounds forthe double click reaction, since organic azide compounds are generallyreactive with diynes. In addition to the aromatic azides, aliphaticazides, azido sugars and azido proteins used as the azide compounds inEXAMPLES later described, there may also be used, for example,azido-amino acids, azido-peptides, azido-nucleic acids, azido-lipids,etc.

The azide compound contains as the substituents (R′ and R″ in generalscheme (I) above) a hydrocarbon group such as a C1-10 alkyl group(methyl, ethyl, propyl, etc.), a C2-10 alkenyl group (ethenyl,1-propenyl, 2-propenyl, etc.), a C2-10 alkynyl group (ethynyl,1-propynyl, 2-propynyl, etc.), a C3-10 cycloalkyl group (cyclopropyl,cyclobutyl, cyclopentyl, etc.), a C3-10 cycloalkenyl group(2-cyclopenten-1-yl, 3-cyclopenten-1-yl, 2-cyclohexen-1-yl, etc.), aC4-10 cycloalkadienyl group (2,4-cyclopentadien-1-yl,2,4-cyclohexadien-1-yl, 2,5-cyclohexadien-1-yl, etc.), a C6-14 arylgroup (phenyl, naphthyl, anthryl, etc.), a C7-13 aralkyl group (benzyl,phenethyl, naphthylmethyl, etc.), a C8-13 arylalkenyl group (styryl,etc.), a C3-10 cycloalkyl-C1-6 alkyl group (cyclohexylmethyl, etc.),etc.; a substituted heteroaromatic ring including a pyridine ring, apyrimidine ring, a pyrazine ring, a furan ring, a thiophen ring, apyrrole ring, an imidazole ring, a triazole ring, an oxazole ring, athiazole ring, etc. and a polycyclic ring system formed by fusing aplurality of these heteroaromatic rings including a C6-14 aryl group;and further contains hydroxy group, amino group, carboxyl group, amidogroup, thioamido group, thiol group, ether group, ester group, sulfogroup, sulfonamido group, etc., in these substituents. In addition tothese compounds, the azide compound further includes protected orunprotected sugar derivatives, nucleic acid derivatives, lipidderivatives, amino acid or peptide derivatives, etc. In thesesubstituents, the carbon number is generally preferably within the rangedescribed above to avoid steric hindrance that might prevent theaddition to the cyclic diyne compound.

More preferably, the azide compound contains as the substituents R′ andR″ a hydrocarbon group such as a C1-6 alkyl group, a C2-6 alkenyl group,a C2-6 alkynyl group, a C6-10 cycloalkyl group, a C6-10 cycloalkenylgroup, a C6-10 cycloalkadienyl group, a C6-12 aryl group, a C7-10aralkyl group, a C8-10 arylalkenyl group, a C6-10 cycloalkyl-C3-6 alkylgroup, etc.

Examples of these preferred azide compounds include methyl azide, ethylazide, propyl azide, azidoacetate, azidoadamantane, phenyl azide and itsderivatives, benzyl azide and its derivatives, cyclohexyl azide, etc.

More specific examples of the preferred azide compound are methyl azide,ethyl azidoacetate, 1-azidoadamantane, phenyl azide, 2-methylphenylazide, 2-isopropylphenyl azide, 2,6-dimethylphenyl azide,2,6-diethylphenyl azide, 2,6-diisopropylphenyl azide,2-t-butyl-6-methylphenyl azide, 2,6-dibromophenyl azide, 4-methoxyphenylazide, 4-trifluoromethylphenyl azide, 3,5-bis (trifluoromethyl)phenylazide, methyl 4-(azidomethyl)benzoate, benzyl azide,4-(azidomethyl)benzyl alcohol, etc.

1D. Cyclic Compound and Triazole Rings

As a result of the double click reaction, the cyclic compound (formulae(1) and (2) as well as (C) and (D) in general scheme (I) describedabove) is produced. In this cyclic compound, two triazole rings areformed by addition of the azide compounds ((B) in general scheme (I)described above). As such, the triazole rings derived from the azidogroups of the azide compounds are both fused to the cyclic skeleton andshare the carbon-carbon double bond sites.

The cyclic compound has the structure corresponding to the cyclic diynecompound and azide compounds employed in the double click reaction. Morespecifically, substituents R¹ and R² are introduced into the triazolerings as shown in formulae (1-1) to (2-3) described below. Furthermore,in the cyclic diyne compound used in the reaction, when the two benzenerings fused to the cyclic skeleton contain no substituent, the benzenering in the cyclic compound contain no substituent (cf., formulae (1-1)and (2-1) described below) and when the two benzene rings containsubstituents R³ and R⁴, the cyclic compound contains the correspondingsubstituents R³ and R⁴ as well (cf., formulae (1-2) and (2-2) describedbelow). Each symbol n in formulae (1-2) and (2-2) described belowindependently represents an integer of 0 to 4. Furthermore, where ringsA and B other than the benzene ring are fused to the cyclic diynecompound, the corresponding rings A and B are contained also in thecyclic compound formed (cf., formulae (1-3) and (2-3) described below).

As is clear from the foregoing, both the cyclic compound containing noneof the substituents R¹ to R⁴ and the cyclic compound having a part orall of substituents R¹ to R⁴ can be used in the double click reaction.The substituents R¹ and R² in formulae (1-1), (2-1), (1-2), (2-2), (1-3)and (2-3) described above are the same as R′ and R″ in general scheme(I) above and the substituents R³ and R⁴ are the same as R in generalscheme (I).

Specific examples of preferred cyclic compounds include those formed bythe double click reaction of the cyclic diyne compound with the azidecompounds which are described above as being preferred. Morespecifically, the cyclic compounds are the following compounds, i.e.;

-   1,8-dihydro-1,8-dimethyldibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d]bis([1,2,3]triazole),-   1,10-dihydro-1,10-dimethyldibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,8-bis(ethoxycarbonylmethyl)-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d]bis([1,2,3]triazole),-   1,10-bis(ethoxycarbonylmethyl)-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d]bis([1,2,3]triazole),-   1,8-diadamantyl-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,10-diadamantyl-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d]bis([1,2,3]triazole),-   1,8-dihydro-1,8-diphenyldibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,10-dihydro-1,10-diphenyldibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,8-dihydro-1,8-bis(2-methylphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,10-dihydro-1,10-bis(2-methylphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   −1,8-dihydro-1,8-bis(2-isopropylphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,10-dihydro-1,10-bis(2-isopropylphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,8-bis(2,6-dimethylphenyl)-1,8-dihydro    dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,10-bis(2,6-dimethylphenyl)-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,8-bis(2,6-diethylphenyl)-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,10-bis(2,6-diethylphenyl)-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,8-bis(2,6-diisopropylphenyl)-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,10-bis(2,6-diisopropylphenyl)-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,8-bis(2-t-butyl-6-methylphenyl)-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,10-bis(2-t-butyl-6-methylphenyl)-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,8-bis(2,6-dibromophenyl)-1,8-dihydro    dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d]bis([1,2,3]triazole),-   1,10-bis(2,6-dibromophenyl)-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,8-dihydro-1,8-bis(4-methoxyphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,10-dihydro-1,10-bis(4-methoxyphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,8-dihydro-1,8-bis(4-trifluoromethylphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,10-dihydro-1,10-bis(4-trifluoromethylphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,8-bis[3,5-bis(trifluoromethyl)phenyl]-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,10-bis[3,5-bis(trifluoromethyl)phenyl]-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,8-dihydro-1,8-bis[4-(methoxycarbonyl)benzyl]dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,10-dihydro-1,10-bis[4-(methoxycarbonyl)benzyl]dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,8-dibenzyl-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,10-dibenzyl-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,8-dihydro-1,8-bis[4-(hydroxymethyl)benzyl]dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),-   1,10-dihydro-1,10-bis[4-(hydroxymethyl)benzyl]dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole),    and so on.

In the cyclic compounds formed by the double click reaction, although itvaries in accordance with the sizes of substituents R¹ and R², the transforms (shown in (C) of the general scheme (I) and formulae (1-1) to(1-3)) which are thermodynamically stable are contained in a higherproportion than cis forms (shown in (D) of the general scheme (I) andformulae (2-1) to (2-3)).

2. Modification of Biomolecules by Double Click Reaction

In the method for modifying a biomolecule according to the presentinvention, the double click reaction (SPDC reaction) is used. That is,the azido group of the azide compound as a probe and the azido grouppreviously introduced into the biomolecule are added and ligated to eachof the two carbon-carbon triple bond sites in the cyclic diyne compoundto form the cyclic compound. The substituent derived from the azidecompound as the probe is incorporated into the cyclic compound thusproduced which contains the biomolecule.

Double click reaction is carried out by ligating an azide compoundhaving the function as a molecular imaging probe, e.g., a fluorescenceprobe, a probe for positron emission tomography (PET), etc., to anazido-protein, an azido-glycoconjugate, etc., which are previouslyprepared. Thus, biomolecules including proteins, glycoconjugates on themembrane surface of living cells, etc. can be modified and labeled whilemaintaining their functions. More specifically, details are described inEXAMPLES 21 and 22 below.

In the double click reaction, the azide compound as the probe can alsobe added sequentially after a biomolecule is added to the cyclic diynecompound. In this sequential double click reaction, it is preferred toremove the unreacted cyclic diyne compound remained after the additionof biomolecule and then add the azide compound only to thebiomolecule-added cyclic diyne compound. On the other hand, while thebiomolecule and the azide compounds are co-present, they can be added tothe cyclic diyne compound. In this case, the biomolecule and the azidecompounds can be ligated in one step via the cyclic diyne compound,which enables efficient chemical modification.

2A. Biomolecule

The biomolecules that can be modified according to the present inventioninclude previously azido-incorporated proteins (including an enzyme,antibody, receptor, hormone, binding factor, ligand, cytoskeletalprotein, etc.), sugars, nucleic acids, and conjugated molecules thereof,etc. In addition, the biomolecules further include not only thoseprepared in vitro but also biomolecules located inside, outside and onthe surface of living cells.

Examples of the biomolecules are a variety of biomolecules, includingcytoskeletal proteins such as actin, tubulin, vimentin, lamin, etc., andfunctional proteins bound thereto such as actinin, cofilin, profilin,Arp2/3, zyxin, MRTF (MAL), etc., motor proteins such as myosin, dynein,kinesin, ATP synthase, etc., adhesion proteins such as cadherin,integrin, etc., extracellular matrix proteins such as collagen,fibronectin, tenascin, biglycan, syndecan, fibrinogen, thrombin, fibrin,laminin, entactin, elastin, fasciclin, periostin, beta ig-h3, versican,decorin, etc., and proteins bound to extracellular matrix(transglutaminase, lysyl oxidase, collagen C-terminal cleavage enzyme,matrix metalloprotease, collagen receptor, etc.), etc., peptides(collagelin, etc.), sugars (hyaluronic acid, etc.), intercellularadhesion complex-associated proteins such as FAK, p130CAS, talin,vinculin, Rap, etc., heat shock proteins, endoplasmic reticulumchaperone proteins such as calreticulin, calnexin, etc., Golgi proteincomponents such as GM130, etc., mitochondrial protein components such ascytochrome complex, etc., cell growth or cell transfer-inducingsecretory proteins such as proteasome protein components, growth factorreceptor proteins, Toll-like receptors, a group of signaling proteinssuch as STAT, MAPK, Ras, Rho, Rac, etc., epidermal growth factor EGF,fibroblast growth factor FGF, platelet-derived growth factor PDGF,neurotrophic factor, insulin, insulin-like growth factor, vascularendothelial growth factor VEGF, stem cell factor, TGFIβ, etc.,inflammatory cytokines such as TNFα, etc., neurotransmitters such asserotonin, adrenaline, etc., hormones such as parathyroid hormone,inhibin, calcitonin, gonadotropic hormone, melatonin, insulin,prolactin, thyroid stimulating hormone, antidiuretic hormone,epinephrine, norepinephrine, androgen, estrogen, corticoid, etc.,transcription activators such as NFκB, c-fos, c-jun, SRF, heat shockfactor HSF, hypoxia-inducible factor HIF, sterol regulatoryelement-binding protein SREBP, Hox family, Sox family, c-myc, c-myb,etc., transcription factor complex protein components such as RNApolymerase, etc., spliceosomal complex protein components such asSAP130, etc., ribosome complex protein components such as S6, etc.,DNA-binding proteins such as nuclear pore complex protein components,telomerase, histone, etc., marker proteins such as annexin Vspecifically bound to apoptotic cells, etc., antibody proteins,luminescent proteins such as aequorin, obelin, clytin, mitrocomin, etc.,fluorescent proteins such as green fluorescent protein (GFP), bluefluorescent protein (BF), fluorescent protein (RFP), etc., luciferasessuch as firefly luciferase, Vargula luciferase, Renilla luciferase,Gaussia luciferase, Oplophorus luciferase, BFP, etc., ligand covalentproteins such as HaloTag proteins previously specifically boundcovalently with a ligand having an azido group, SNAP-tag proteins,CLIP-tag proteins, etc., low molecular ligand non-covalent proteins suchas avidin, streptavidin, dehydrofolate reductase, tetracysteine motif(—CCXXCC—: X is an optional amino acid) peptide sequence-containingproteins, tetraserine motif (—SSXXSS—: X is an optional amino acid)peptide sequence-containing proteins, etc. The biomolecules furtherinclude a variety of molecules including kinases such as protein kinaseC, protein kinase A, calmodulin, DYRK, p70S6K, Cik, receptor typekinases (fibroblast growth factor receptor, neurotrophic factorreceptor, fibroblast growth factor receptor, insulin receptor,insulin-like growth factor receptor, vascular endothelial growth factorreceptor, stem cell factor receptor, etc.), mTOR complex, GSK3, MAPkinase, Mos/Raf kinase, cdc2, etc., phosphatases such as calcineurin,lipid phosphatase PTEN, histidine phosphatase, serine/threonine-specificphosphatase, tyrosine-specific phosphatase, acidic phosphatase, alkalinephosphatase, etc., and all variants thereof, proteins previouslyincorporated with an azido group and obtained by fusing a plurality ofthe proteins above via a suitable linker, respectively, peptides andproteins prepared using non-natural amino acids containing an azidogroup such as azidotyrosine, azidophenylalanine, azidoalanine,azidohomoalanine, etc., glycoconjugates at the cell membrane surfaceincluding derivatives of previously azido-incorporated sialic acid,glucose, glucosamine, mannose, mannosamine, galactose, galactosamine,ribose, deoxyribose, etc., nucleotides such as previouslyazido-incorporated oligonucleotides, deoxynucleotides,morpholinonucleotides, etc., nucleotides and deoxynucleotidessynthesized from nucleic acids such as azidoadenosine, azidoguanosine,azidothymidine, azidocytosine, azidouracil, etc., lipids such aspreviously azido-incorporated diacylglycerols, ceramides,sphingophospholipids, glycerophospholipids, sphingoglycolipids,glyceroglycolipids, sulfo lipids, fatty acids, terpenoids, steroids,carotenoids, etc., and biological culture cells, etc., all of thenatural biomolecules described above previously incorporated with anazido group that are chemically modified at random or site-specificallyby photo-cross-linking or with a highly reactive functional lowmolecular ligand such as FITC, etc.

The biomolecules further include artificial biomolecules such asproteins artificially prepared using Escherichia coli, insect cells,yeast, etc., proteins artificially synthesized from amino acids in vivo.These biomolecules can all be labeled with probes.

Hereinafter, Examples are explained. However, the present invention isnot limited to those Examples.

Method for Synthesis of Cyclic Diyne Compound

Diyne 3 (sym-dibenzo-1,5-cyclooctadiene-3,7-diyne) used as the cyclicdiyne compound in EXAMPLES later described can be easily synthesized bythe methods described in, e.g., Non-Patent Documents (a) H. N.C. Wong,P. J. Garratt, F. Sondheimer, J. Am. Chem. Soc. 1974, 96, 5604-5605; b)A. Orita, D. Hasegawa, T. Nakano, J. Otera, Chem. Eur. J. 2002, 8,2000-2004; and c) S. Chaffins, M. Brcttreich, F. Wudl, Synthesis 2002,1191-1194).

In the synthesis of each compound, flash column chromatography wasperformed using silica-gel (37563-85 manufactured by Kanto Chemical Co.,Inc., Silica Gel 60 N (spherical, neutral), particle size of 40-50 p.m),or 37565-85 manufactured by Kanto Chemical Co., Inc., Silica Gel 60 N(spherical, neutral), particle size of 63-210 μm).

Thin-layer chromatography (TLC) was performed using a glass platepreviously coated with silica-gel (1.05715 manufactured by Merck Inc.,Silica Gel 60 F₂₅₄).

Measurement of Physical Properties of Compound

Structural analyses and physical properties of the compounds produced inEXAMPLES later described were determined as follows.

Melting point (Mp) was measured using a MP-J3 Micro Melting PointMeasuring Apparatus manufactured by YANACO New Science Inc. (uncorrecteddata).

¹H and ¹³C NMR (nuclear magnetic resonance) spectra were measured at 300MHz and 75.5 MHz, respectively, using a Mercury 300 NMR Spectrometermanufactured by Varian Inc., or at 500 MHz and 126 MHz, respectively,using an AVANCE 500 NMR Spectrometer manufactured by Bruker Corp. CDCl₃,DMSO-d₆ or CD₃OD (all manufactured by CIL Inc.) was used as a solventfor NMR spectrometry.

Chemical shifts (δ) are given in relative values downfield fromtetramethylsilane ((CH₃)₄Si) (δ 0 ppm for ¹H NMR measured in CDCl₃) orthe solvent peak (δ 2.49 ppm for ¹H NMR in DMSO-d₆; δ 3.30 ppm for ¹HNMR in CD₃OD; and δ 77.0 ppm for ¹³C NMR in CDCl₃) as an internalreference with coupling constants (J) in Hz. The abbreviations s, d, t,q, m and br later described signify singlet, doublet, triplet, quartet,multiplet and broad, respectively.

Infrared (IR) spectra were measured by a diffuse reflectance method onan IRPrestige-21 Fourier Transform Infrared Spectrophotometer attachedwith DRS-8000A Diffuse Reflectance Accessory, manufactured by SHIMADZUCorp.

Ultraviolet (UV) absorbance spectra were measured with a UV-3100Ultraviolet Visible Infrared Spectrophotometer, manufactured by SHIMADZUCorp. at 25° C. using a quartz cuvette (10 mm light path) under theconditions of a high speed scanning rate.

Fluorescence (FL) spectra were measured with a RF-5300PCspectrofluorophotometer manufactured by SHIMADZU Corp. at 25° C. using aquartz cuvette (10 mm light path) under the conditions of emission andexcitation bandwidth, 3 nm and scan speed, medium.

High-resolution mass spectra (HRMS) were measured by positive fast atombombardment (FAB⁺) method or electron impact ionization (EI) methodusing a JMS-700 mass spectrometer manufactured by JEOL, or by positiveelectrospray ionization (ESI⁺) method using a micrOTOF mass spectrometermanufactured by Bruker Inc. In the FAB⁺ method, m-nitrobenzyl alcohol(NBA) was used as a matrix.

X-Ray crystallography was determined on an Imaging Plate Single CrystalX-ray Diffractometer for Structure Analysis manufactured by Rigaku Corp.Crystal structure was determined using a SHELXL-97 program. The crystalstructure data were registered in CSD (Cambridge Crystal StructureDatabases) and are available, upon request, from CambridgeCrystallographic Data Centre: CCDC,www.ccdc.-cam.ac.uk/data_request/cif.

Example 1 Production 1 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (4) above, to a solution of diyne 3(40.0 mg, 200 μmol) in methanol (23.5 mL) was added a solution of benzylazide (4a, commercial product) (63.9 mg, 480 μmol) in methanol (1.5 mL)at room temperature. After stirring for 70 minutes at the sametemperature, the reaction solution was concentrated under reducedpressure using an evaporator. The residue was purified by flash columnchromatography (silica-gel 10 g, dichloromethane only todichloromethane/methanol=6/1) to give two regioisomeric bis-cycloadductsof trans-6a (55.8 mg, 120 μmol, 59.9%) and cis-7a (35.2 mg, 75.4 μmol,37.8%). The geometries of the respective compounds were confirmed byX-ray crystallographical analyses (CCDC 759900 (6a) and CCDC 759902(7a)).

1,8-Dibenzyl-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(6a)

Recrystallization from n-hexane/ethyl acetate gave colorless crystals.The following physical properties of the crystals were measured toconduct structural analysis.

Mp 230-232° C.;

R_(f)=0.26 (hexane/ethyl acetate=1/1);R_(f)=0.69 (dichloromethane/methanol=9/1);

¹H NMR (300 MHz, CDCl₃) δ 5.31 (d, 2H, J=15.3 Hz), 5.50 (d, 2H, J=15.3Hz), 6.94-7.01 (m, 4H), 7.09 (dd, 2H, J=0.8, 7.6 Hz), 7.22-7.30 (m, 6H),7.40 (ddd, 2H, J=1.3, 7.6, 7.6 Hz), 7.52 (ddd, 2H, J=1.3, 7.6, 7.6 Hz),7.71 (dd, 2H, J=0.8, 7.6 Hz);

¹³C NMR (75.5 MHz, CDCl₃) δ 52.1 (2C), 126.4 (2C), 127.0 (4C), 128.2(2C), 128.7 (2C), 128.8 (4C), 129.9 (2C), 130.2 (2C), 131.2 (2C), 132.6(2C), 134.9 (2C), 135.1 (2C), 145.0 (2C);

IR (KBr, cm⁻¹) 706, 731, 764, 910, 984, 1028, 1215, 1250, 1350, 1454,1497, 1514, 3063;

HRMS (ESI⁺) m/z 467.1984 ([M+H]⁺, C₃₀H₂₃N₆ ⁺ Calcd. 467.1979).

1,10-Dibenzyl-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(7a)

Recrystallization from n-hexane/ethyl acetate gave colorless crystals.The following physical properties of the crystals were measured toconduct structural analysis.

Mp 274-277° C.;

R_(f)=0.26 (hexane/ethyl acetate=1/1);R_(f)=0.44 (dichloromethane/methanol=9/1);

¹H NMR (300 MHz, CDCl₃) δ 4.89 (d, 2H, J=15.5 Hz), 5.31 (d, 2H, J=15.5Hz), 6.94-7.04 (m, 4H), 7.04-7.14 (m, 2H), 7.24-7.36 (m, 6H), 7.36-7.45(m, 2H), 7.45-7.54 (m, 2H), 7.62-7.72 (m, 2H);

¹³C NMR (75.5 MHz, CDCl₃) δ 52.0 (2C), 127.2 (4C), 127.9 (2C), 128.3(2C), 128.8 (4C), 129.1 (2C), 129.8 (2C), 130.3 (2C), 130.7 (2C), 130.9(2C), 133.6 (2C), 135.3 (2C), 146.2 (2C);

IR (KBr, cm⁻¹) 698, 729, 766, 910, 984, 1028, 1211, 1246, 1344, 1427,1454, 1497, 1516, 3061;

HRMS (EST⁺) m/z 467.1982 ([M+H]⁺, C₃₀H₂₃N₆ ⁺ Calcd. 467.1979).

Example 2 Production 2 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (5) above, to a solution of diyne 3(40.0 mg, 200 μmol) in methanol (23.5 mL) was added a solution of benzylazide (4a) (26.6 mg, 200 μmol) in methanol (1.5 mL) at room temperature.After stirring for 24 hours at the same temperature, the reactionsolution was concentrated under reduced pressure using an evaporator.The residue was purified by flash column chromatography (silica-gel 10g, dichloromethane only to dichloromethane/methanol=6/1) to give tworegioisomeric bis-cycloadducts of trans-6a (22.0 mg, 47.2 μmol, 23.6%)and cis-7a (19.0 mg, 40.7 μmol, 20.4%) along with recovery of startingdiyne 3 (22.7 mg, 113 μmol, 56.8%).

In the SPDC reaction of EXAMPLE 1 described above, the mono-cycloadduct5a (cf., reaction scheme (4) above), a presumable monoyne intermediate,was neither detected nor isolated. Furthermore, as in the SPDC reactionof EXAMPLE 2, the mono-cycloadduct 5a was not obtained even by reactionof diyne 3 with an equimolar amount of benzyl azide (4a) butbis-cycloadducts 6a and 7a and diyne 3 were only recovered. The combinedyield of bis-cycloadducts 6a and 7a and recovered diyne 3 was 100% basedon the starting diyne 3. These results indicate that the intermediate 5ais more reactive toward benzyl azide 4a than the starting diyne 3. Assuch, the higher reactivity of the intermediate in the SPDC reactionthan the starting diyne was also supported by calculated results of theactivation energy, etc. of the reactions later described.

As such, two azide compounds are added and ligated to the cyclic diynecompound to form the cyclic skeleton of an 8-membered ring and the two1,2,3-triazole ring sharing the carbon-carbon double bond sites with thecyclic skeleton. Diyne 3 has a strain, is stable and easily handled andthus suitable as the substrate for the SPDC reaction. As shown informula (3) above, the cyclic diyne compound having a substituent suchas a hydrocarbon group(s) on either one or both of the two benzene ringsof diyne 3 can be used instead of diyne 3.

Example 3 Production 3 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (6) above, to a solution of diyne 3(40.0 mg, 200 μmol) in methanol (23.5 mL) was added a solution of ethylazidoacetate (4b, commercial product) (55.0 μL, 480 μmol) in methanol(1.5 mL) at room temperature. After stirring for 60 minutes at the sametemperature, the reaction solution was concentrated under reducedpressure using an evaporator. The residue was purified by flash columnchromatography (silica-gel 10 g, hexane/ethyl acetate=1/1) to give tworegioisomeric bis-cycloadducts of trans-6b (41.9 mg, 91.5 μmol, 45.8%)and cis-7b (38.8 mg, 84.7 μmol, 42.4%). The geometries of the respectivecompounds were confirmed by X-ray crystallographical analyses (CCDC759901 (6b) and CCDC 759903 (7b)).

1,8-Bis(ethoxycarbonylmethyl)-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(6b)

Recrystallization from n-hexane/ethyl acetate gave colorless crystals.The following physical properties of the crystals were measured toconduct structural analysis.

Mp 204-205° C.;

R_(f)=0.55 (hexane/ethyl acetate=1/3);

¹H NMR (300 MHz, CDCl₃) δ 1.23 (t, 6H, J=7.1 Hz), 4.10-4.34 (m, 4H),4.87 (d, 2H, J=17.4 Hz), 5.09 (d, 2H, J=17.4 Hz), 7.31 (d, 2H, J=7.1Hz), 7.48 (dd, 2H, J=7.1, 7.1 Hz), 7.57 (dd, 2H, J=7.1, 7.1 Hz), 7.78(d, 2H, J=7.1 Hz);

¹³C NMR (75.5 MHz, CDCl₃) δ 13.8 (2C), 49.4 (2C), 62.3 (2C), 125.7 (2C),129.0 (2C), 129.1 (2C), 130.3 (2C), 131.5 (2C), 132.4 (2C), 135.3 (2C),144.4 (2C), 166.3 (2C);

IR (KBr, cm⁻) 737, 766, 779, 876, 910, 986, 1022, 1134, 1161, 1211,1256, 1348, 1364, 1418, 1474, 1516, 1748, 2982;

HRMS (ESI⁺) m/z 459.1788 ([M+H]⁺, C₂₄H₂₃N₆O₄ ⁺ Calcd. 459.1775).

1,10-Bis(ethoxycarbonylmethyl)-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(7b)

Recrystallization from n-hexane/ethyl acetate gave colorless crystals.The following physical properties of the crystals were measured toconduct structural analysis.

Mp 214-217° C.;

R_(f)=0.38 (hexane/ethyl acetate=1/3);

¹H NMR (300 MHz, CDCl₃) δ 1.26 (t, 6H, J=7.1 Hz), 4.21 (q, 4H, J=7.1Hz), 5.03 (d, 2H, J=17.4 Hz), 5.13 (d, 2H, J=17.4 Hz), 7.35-7.44 (m,2H), 7.47-7.59 (m, 4H), 7.66-7.76 (m, 2H);

¹³C NMR (75.5 MHz, CDCl₃) δ 13.9 (2C), 49.3 (2C), 62.2 (2C), 128.3 (2C),129.0 (2C), 129.8 (2C), 130.19 (2C), 130.22 (2C), 130.8 (2C), 134.5(2C), 145.6 (2C), 166.5 (2C);

IR (KBr, cm⁻¹) 733, 768, 799, 876, 912, 986, 1020, 1132, 1161, 1211,1250, 1346, 1362, 1414, 1474, 1518, 1748, 2984;

HRMS (ESI⁺) m/z 459.1783 ([M+H]⁺, C₂₄H₂₃N₆O₄ ⁺ Calcd. 459.1775).

Example 4 Production 4 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (7) above, to a solution of diyne 3(40.0 mg, 200 μmol) in methanol (23.5 mL) was added a solution of phenylazide (4c, synthesized by the method described in Non-Patent Document N.D. Obushak, N. T. Pokhodylo, N. I. Pidlypnyi, V. S. Matiichuk, Russ. J.Org. Chem. 2008, 44, 1522-1527) (57.2 mg, 480 1=01) in methanol (1.5 mL)at room temperature. After stirring for 90 minutes at the sametemperature, the reaction solution was concentrated under reducedpressure using an evaporator. The residue was purified by flash columnchromatography (silica-gel 10 g, hexane/ethyl acetate=4/1) to give tworegioisomeric bis-cycloadducts of trans-6c (36.5 mg, 83.2 μmol, 41.7%)and cis-7c (46.1 mg, 105 μmol, 52.6%). The geometries of the respectivecompounds were confirmed by X-ray crystallographical analyses (CCDC759898 (6c) and CCDC 759905 (7c)).

1,8-Dihydro-1,8-diphenyldibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(6c)

Recrystallization from n-hexane/dichloromethane gave colorless crystals.The following physical properties of the crystals were measured toconduct structural analysis.

Mp>300° C.;

R_(f)=0.23 (hexane/ethyl acetate=3/1);

¹H NMR (300 MHz, CDCl₃) δ 6.84 (dd, 2H, J=1.2, 7.8 Hz), 7.21 (ddd, 2H,J=1.2, 7.8, 7.8 Hz), 7.29-7.43 (m, 10H), 7.48 (ddd, 2H, J=1.2, 7.8, 7.8Hz), 7.83 (dd, 2H, J=1.2, 7.8 Hz);

¹³C NMR (75.5 MHz, CDCl₃) δ 125.0 (4C), 126.7 (2C), 128.8 (2C), 129.1(2C), 129.3 (4C), 130.0 (2C), 130.8 (2C), 131.3 (2C), 131.9 (2C), 134.2(2C), 135.9 (2C), 145.8 (2C);

IR (KBr, cm⁻¹) 692, 734, 768, 997, 1265, 1361, 1497, 1512, 1595;

HRMS (ESI⁺) m/z 439.1667 ([M+H]⁺, C₂₈H₁₉N₆ ⁺ Calcd. 439.1666).

1,10-Dihydro-1,10-diphenyldibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole) (7c)

Recrystallization from n-hexane/dichloromethane gave colorless crystals.The following physical properties of the crystals were measured toconduct structural analysis.

Mp>300° C.;

R_(f)=0.13 (hexane/ethyl acetate=3/1);

¹H NMR (300 MHz, CDCl₃) δ 6.87-6.95 (m, 2H), 7.12-7.21 (m, 2H),7.39-7.51 (m, 10H), 7.52-7.62 (m, 2H), 7.77-7.85 (m, 2H);

¹³C NMR (75.5 MHz, CDCl₃) δ 126.1 (4C), 127.4 (2C), 129.4 (4C), 129.6(2C), 129.8 (2C), 130.3 (2C), 130.6 (2C), 131.4 (2C), 131.6 (2C), 134.1(2C), 135.8 (2C), 145.1 (2C);

IR (KBr, cm⁻¹) 527, 608, 687, 734, 762, 997, 1069, 1132, 1175, 1263,1358, 1427, 1476, 1497, 1514, 1595;

HRMS (ESI⁺) m/z 439.1670 ([M+H]⁺, C₂₈H₁₉N₆ ⁺ Calcd. 439.1666).

Example 5 Production 5 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (8) above, to a solution of diyne 3(40.0 mg, 200 μmmol) in methanol (25.0 mL) was added 1-azidoadamantane(4 g) (85.1 mg, 480 μmol) at room temperature. After stirring for 24hours at room temperature, the reaction solution was concentrated underreduced pressure. The residue was purified by thin-layer chromatography(dichloromethane/methanol=49/1) to give trans-6 g (27.5 mg, 49.6 μmol,24.8%) and cis-7 g (77.9 mg, 140 μmol, 70.3%). The geometries of therespective compounds were confirmed by X-ray crystallographical analyses(CCDC 810930 (6 g) and CCDC 810837 (7 g)).

1,8-Diadamantyl-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole) (6 g)

The following physical properties of the colorless crystals obtainedwere measured to perform structural analysis.

Mp 240-242° C.;

R_(f)=0.79 (dichloromethane/methanol=9/1);

¹H NMR (500 MHz, CDCl₃) δ 1.56-1.65 (m, 12H), 2.02-2.11 (m, 12H),2.21-2.25 (m, 6H), 7.32-7.38 (m, 4H), 7.47 (ddd, 2H, J=1.5, 7.5, 7.5Hz), 7.64 (d, 2H, J=7.5 Hz);

¹³C NMR (126 MHz, CDCl₃) 29.7 (6C), 35.7 (6C), 42.7 (6C), 63.9 (2C),127.6 (2C), 129.6 (2C), 129.8 (2C), 130.5 (2C), 130.9 (2C), 132.5 (2C),134.3 (2C), 146.5 (2C);

IR (KBr, cm⁻¹) 731, 764, 777, 839, 912, 1011, 1101, 1125, 1260, 1306,1323, 1358, 1452, 2853, 2909;

HRMS (ESI⁺) m/z 555.3237 ([M+H]⁺, C₃₆H₃₉N₆ ⁺ requires 555.3231).

1,10-Diadamantyl-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(7 g)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

Mp>300° C.;

R_(f)=0.57 (dichloromethane/methanol=9/1);

¹H NMR (500 MHz, CDCl₃) δ 1.68 (br d, 6H, J=12.0 Hz), 1.74 (br d, 6H,J=12.0 Hz), 2.18 (br s, 6H), 2.34 (br s, 12H), 7.31-7.35 (m, 2H),7.36-7.43 (m, 4H), 7.58-7.63 (m, 2H);

¹³C NMR (126 MHz, CDCl₃) δ 29.8 (6C), 35.8 (6C), 44.0 (6C), 63.9 (2C),128.4 (2C), 128.7 (2C), 130.2 (2C), 130.8 (2C), 131.1 (2C), 132.3 (2C),132.9 (2C), 147.2 (2C); IR (KBr, cm⁻¹) 737, 766, 908, 1011, 1101, 1308,1321, 1452, 2855, 2914;

HRMS (ESI⁺) m/z 555.3257 ([M+H]⁺, C₃₆H₃₉N₆ ⁺ requires 555.3231).

Example 6 Production 6 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (9) above, to a solution of diyne 3(10.0 mg, 50.0 μmol) in methanol (5.00 mL) was added a solution of2,6-diisopropylphenyl azide (4 h) (24.4 mg, 120 μmol in methanol (1.25mL) at room temperature. After stirring for 30 minutes at roomtemperature, the reaction solution was concentrated under reducedpressure. The residue was purified by flash column chromatography(silica-gel 10 g, dichloromethane/methanol=100/1) to give a mixture oftrans-6 h and cis-7 h (28.9 mg, 47.6 μmol, 95.4%). The ratio of thetrans-6 h to cis-7 h was determined based on the ¹H NMR spectrum. Thegeometries of the respective compounds were confirmed by X-raycrystallographical analyses (CCDC 810931 (6 h) and CCDC 810838 (7 h)).

1,8-Bis(2,6-diisopropylphenyl)-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole) (6 h)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

Mp 273-275° C.;

R_(f)=0.65 (dichloromethane/methanol=49/1);

¹H NMR (500 MHz, CDCl₃) δ 0.73 (d, 6H, J=7.0 Hz), 0.78 (d, 6H, J=7.0Hz), 1.410 (d, 6H, J=6.5 Hz), 1.413 (d, 6H, J=6.5 Hz), 1.96 (qq, 2H,J=7.0, 7.0 Hz), 2.63 (qq, 2H, J=6.5, 6.5 Hz), 6.87 (d, 2H, J=7.5 Hz),7.06 (d, 2H, J=7.5 Hz), 7.17 (ddd, 2H, J=1.0, 7.8, 7.8 Hz), 7.35 (dd,2H, J=1.0, 7.8 Hz), 7.38-7.45 (m, 4H), 7.81 (d, 2H, J=7.5 Hz);

¹³C NMR (126 MHz, CDCl₃) δ 22.3 (2C), 22.8 (2C), 24.4 (2C), 26.2 (2C),28.3 (2C), 29.2 (2C), 123.85 (2C), 123.92 (2C), 126.3 (2C), 128.4 (2C),129.6 (2C), 129.7 (2C), 130.7 (2C), 131.5 (4C), 132.5 (2C), 135.9 (2C),144.1 (2C), 145.6 (2C), 146.9 (2C);

IR (KBr, cm⁻¹) 733, 756, 768, 995, 1362, 1470, 2868, 2930, 2965;

HRMS (ESI⁺) m/z 607.3525 ([M+H]⁻¹, C₄₀H₄₃N₆ ⁺ requires 607.3544);C₄₀H₄₂N₆: Calcd.: C, 79.17; H, 6.98; N, 13.85%; Found: C, 79.09; H,6.99; N, 13.59%.

1,10-Bis(2,6-diisopropylphenyl)-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(7 h)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

R_(f)=0.40 (dichloromethane/methanol=49/1);

¹H NMR (500 MHz, CDCl₃) δ 0.42 (d, 6H, J=6.8 Hz), 0.98 (d, 6H, J=6.8Hz), 1.29 (d, 6H, J=6.8 Hz), 1.32 (d, 6H, J=6.8 Hz), 2.07 (dq, 2H,J=6.8, 6.8 Hz), 2.21 (dq, 2H, J=6.8, 6.8 Hz), 6.93 (dd, 2H, J=3.5, 5.5Hz), 7.03 (dd, 211, J=3.5, 5.5 Hz), 7.12 (d, 2H, J=7.5 Hz), 7.33 (d, 2H,J=7.5 Hz), 7.45 (dd, 2H, J=7.5, 7.5 Hz), 7.55 (dd, 2H, J=3.5, 5.5 Hz),7.76 (dd, 2H, J=3.5, 5.5 Hz);

¹³C NMR (126 MHz, CDCl₃) δ 21.8 (2C), 22.5 (2C), 25.5 (2C), 26.2 (2C),29.0 (2C), 29.3 (2C), 124.26 (2C), 124.34 (2C), 127.7 (2C), 128.8 (2C),129.2 (2C), 130.9 (2C), 131.00 (2C), 131.02 (2C), 131.1 (2C), 132.3(2C), 135.7 (2C), 145.7 (2C), 146.2 (2C), 147.9 (2C);

IR (KBr, cm⁻¹) 733, 764, 991, 1277, 1354, 1456, 1724, 2868, 2928, 2964;

HRMS (ESI⁺) m/z 629.3348 ([M+Na]⁺, C₄₀H₄₂N₆Na⁺ requires 629.3363).

Example 7 Production 7 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (10) above, to a solution of diyne 3(40.0 mg, 200 μmol) in methanol (22.0 mL) was added a solution of4-methoxyphenyl azide (4i) (71.6 mg, 480 μmol) in methanol (3.00 mL) atroom temperature. After stirring for 20 minutes at room temperature, thereaction solution was concentrated under reduced pressure. The residuewas purified by flash column chromatography (silica-gel 10 g,dichloromethane only to dichloromethane/methanol=49/1) to give a mixtureof trans-6i and cis-7i (88.8 mg, 178 μmol, 89.2%). The ratio of trans-6iand cis-7i was determined based on the ¹H NMR spectrum. The geometriesof the respective compounds were confirmed by X-ray crystallographicalanalyses (CCDC 810932 (6i) and CCDC 810839 (7i)).

1,8-Dihydro-1,8-bis(4-methoxyphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(6i)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

Mp 250-251° C.;

R_(f)=0.47 (n-hexane/ethyl acetate/dichloromethane/toluene=1/1/1/1);

¹H NMR (500 MHz, CDCl₃) δ 3.82 (s, 6H), 6.83-6.88 (m, 6H), 7.19-7.24 (m,6H), 7.47 (ddd, 2H, J=1.0, 7.5, 7.5 Hz), 7.81 (d, 2H, J=7.5 Hz);

¹³C NMR (126 MHz, CDCl₃) δ 55.5 (2C), 114.4 (4C), 126.4 (4C), 126.8(2C), 128.8 (2C), 129.1 (2C), 129.9 (2C), 130.8 (2C), 131.2 (2C), 132.1(2C), 134.2 (2C), 145.7 (2C), 159.9 (2C);

IR (KBr, cm⁺) 538, 592, 733, 764, 833, 910, 993, 1028, 1053, 1103, 1115,1169, 1182, 1252, 1302, 1443, 1464, 1514, 1589, 1609;

HRMS (ESI⁺) m/z 499.1886 ([M+H]⁺, C₃₀H₂₃N₆O₂ ⁺ requires 499.1877);

C₃₀H₂₂N₆O₂: Calcd.: C, 72.28; H, 4.45; N, 16.86%; Found: C, 72.09; H,4.32; N, 16.60%.

1,10-Dihydro-1,10-bis(4-methoxyphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(7i)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

Mp>300° C.;

R_(f)=0.38 (n-hexane/ethyl acetate/dichloromethane/toluene=1/1/1/1);

¹H NMR (500 MHz, CDCl₃) δ 3.87 (s, 6H), 6.89-6.95 (m, 2H), 6.94 (AA′BB′,4H), 7.13-7.19 (m, 2H), 7.31 (AA′BB′, 4H), 7.52-7.57 (m, 2H), 7.76-7.82(m, 2H);

¹³C NMR (126 MHz, CDCl₃) δ 55.6 (2C), 114.5 (4C), 126.5 (4C), 128.4(2C), 129.2 (2C), 129.3 (2C), 129.4 (2C), 130.5 (2C), 130.7 (2C), 131.6(2C), 133.5 (2C), 146.4 (2C), 160.1 (2C);

IR (KBr, cm⁻¹) 532, 594, 733, 762, 775, 831, 995, 1020, 1036, 1256,1306, 1516, 1611;

HRMS (ESI⁺) m/z 499.1876 ([M+H]⁺, C₃₀H₂₂N₆O₂ ⁺ requires 499.1877);

C₃₀H₂₂N₆O₂: Calcd.: C, 72.28; H, 4.45; N, 16.86%; Found: C, 72.14; H,4.38; N, 16.81%.

Example 8 Production 8 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (11) above, to a solution of diyne 3(40.0 mg, 200 μmol) in methanol (22.0 mL) was added a solution of4-trifluoromethylphenyl azide (4j) (89.8 mg, 480 μmol) in methanol (3.00mL) at room temperature. After stirring for 24 hours at roomtemperature, the reaction solution was concentrated under reducedpressure. The residue was purified by thin-layer chromatography(n-hexane/ethyl acetate=9/1) to give a mixture of trans-6j and cis-7j(111 mg, 193 μmol, 96.7%). The ratio of trans-6j and cis-7j wasdetermined based on the ¹H NMR spectrum.

1,8-Dihydro-1,8-bis(4-trifluoromethylphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(6j)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

Mp 270-272° C.;

R_(f)=0.19 (n-hexane/ethyl acetate=9/1);

¹H NMR (500 MHz, CDCl₃) δ 6.85 (dd, 2H, J=1.0, 7.5 Hz), 7.28 (ddd, 2H,J=1.0, 7.5, 7.5 Hz), 7.45 (d, 4H, J=8.5 Hz), 7.55 (ddd, 2H, J=1.0, 7.5,7.5 Hz), 7.66 (d, 4H, J=8.5 Hz), 7.86 (dd, 2H, J=1.0, 7.5 Hz);

¹³C NMR (126 MHz, CDCl₃) δ 123.4 (q, 2C, =273 Hz), 125.0 (4C), 126.2(2C), 126.6 (q, 4C, J³ _(cf)=3.5 Hz), 129.4 (2C), 130.5 (2C), 130.8(2C), 131.1 (q, 2C, J² _(ef)=32.9 Hz), 131.5 (2C), 131.6 (2C), 134.2(2C), 138.6 (2C), 146.1 (2C);

IR (KBr, cm⁻¹) 733, 764, 845, 995, 1043, 1070, 1130, 1171, 1323, 1366,1406, 1520, 1614, 3065;

HRMS (ESI⁺) m/z 575.1399 ([M+H]⁺, C₃₀H₁₇F₆N₆ ⁺ requires 575.1413).

1,10-Dihydro-1,10-bis(4-trifluoromethylphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(7j)

Mp>300° C.;

R_(f)=0.13 (n-hexane/ethyl acetate=9/1);

¹H NMR (500 MHz, CDCl₃) δ 6.91-6.96 (m, 2H), 7.24-7.30 (m, 2H), 7.56 (d,2H, J=8.0 Hz), 7.56-7.61 (m, 4H), 7.78 (d, 2H, J=8.0 Hz), 7.77-7.82 (m,4H);

¹³C NMR (126 MHz, CDCl₃) δ 123.3 (q, 2C, J¹ _(cf)=273 Hz), 124.9 (4C),126.8 (q, 4C, J³ _(cf)=3.5 Hz), 127.8 (2C), 129.7 (2C), 129.9 (2C),130.2 (2C), 130.9 (2C), 131.4 (q, 2C, J² _(cf)=33.3 Hz), 131.9 (2C),133.2 (2C), 138.9 (2C), 147.1 (2C);

IR (KBr, cm⁻¹) 733, 764, 775, 843, 997, 1047, 1063, 1076, 1119, 1169,1329, 1360, 1412, 1522, 1618, 3071;

HRMS (ESI⁺) m/z 575.1412 ([M+H]⁺, C₃₀H₁₇F₆N₆ ⁺ requires 575.1413);

C₃₀H₁₆F₆N₆: Calcd.: C, 62.72; H, 2.81; N, 14.63%; Found: C, 63.01; H,3.06; N, 14.48%.

Example 9 Production 9 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (12) above, to a solution of diyne 3(10.0 mg, 50.0 μmol) in methanol (5.00 mL) was added a solution of2-methylphenyl azide (4k) (16.0 mg, 120 μmol) in methanol (1.25 mL) atroom temperature. After stirring for 24 hours at room temperature, thereaction solution was concentrated under reduced pressure. The residuewas purified by flash column chromatography (silica-gel 10 g,dichloromethane only to dichloromethane/methanol=9/1) to give a mixtureof trans-6k and cis-7k (22.9 mg, 49.1 μmol, 98.3%). The ratio oftrans-6k and cis-7k was determined based on the ¹H NMR spectrum. Thegeometries of the respective compounds were confirmed by X-raycrystallographical analyses (CCDC 810933 (6k) and CCDC 810840 (7k)).

1,8-Dihydro-1,8-bis(2-methylphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(6k)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

Mp>300° C.;

R_(f)=0.36 (dichloromethane/methanol=49/1);

¹H NMR (500 MHz, CDCl₃) δ 1.88 (br s, 6H), 6.86 (br d, 2H, J=7.5 Hz),7.17 (ddd, 2H, J=1.0, 1.0, 7.5 Hz), 7.19-7.37 (m, 8H), 7.43 (ddd, 2H,J=1.0, 7.5, 7.5 Hz), 7.77 (dd, 2H, J=1.0, 7.5 Hz);

¹³C NMR (126 MHz, CDCl₃) δ 17.5 (2C), 126.4 (2C), 126.7 (2C), 127.9(2C), 128.6 (2C), 129.9 (2C), 130.0 (2C), 130.4 (2C), 131.2 (2C), 131.3(2C), 132.1 (2C), 135.09 (2C), 135.10 (2C), 135.8 (2C), 144.8 (2C);

IR (KBr, cm⁻¹) 613, 719, 739, 770, 908, 997, 1267, 1362, 1425, 1466,1497, 1514;

HRMS (ESI⁺) m/z 467.1958 ([M+H]⁺, C₃₀H₂₃N₆ ⁺ requires 467.1979);

C₃₀H₂₂N₆: Calcd.: C, 77.23; H, 4.75; N, 18.01%; Found: C, 77.19; H,4.65; N, 17.80%.

1,10-Dihydro-1,10-bis(2-methylphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(7k)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

Mp>300° C.;

R_(f)=0.22 (dichloromethane/methanol=49/1);

¹H NMR (500 MHz, CDCl₃) δ 2.33 (s, 6H), 6.75-6.80 (m, 2H), 7.01 (d, 2H,J=7.5 Hz), 7.03-7.08 (m, 2H), 7.17 (dd, 2H, J=7.5, 7.5 Hz), 7.37 (dd,2H, J=7.5, 7.5 Hz), 7.40 (d, 2H, J=7.5 Hz), 7.54-7.59 (m, 2H), 7.79-7.84(m, 2H);

¹³C NMR (126 MHz, CDCl₃) δ 18.3 (2C), 126.4 (2C), 127.2 (2C), 127.8(2C), 129.2 (2C), 129.3 (2C), 130.1 (2C), 130.6 (2C), 130.9 (2C), 131.0(2C), 131.6 (2C), 134.3 (2C), 135.2 (2C), 135.7 (2C), 145.9 (2C);

IR (KBr, cm⁻¹) 611, 718, 737, 762, 781, 999, 1117, 1138, 1263, 1287,1356, 1429, 1462, 1473, 1497, 1512;

HRMS (ESI⁺) m/z 467.1979 ([M+H]⁺, C₃₀H₂₃N₆ ⁺ requires 467.1979);

C₃₀H₂₂N₆: Calcd.: C, 77.23; H, 4.75; N, 18.01%; Found: C, 77.22; H,4.68; N, 17.77%.

Example 10 Production 10 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (13) above, to a solution of diyne 3(40.0 mg, 200 μmol) in methanol (22.0 mL) was added a solution of2-isopropylphenyl azide (41) (77.4 mg, 480 μmol) in methanol (3.0 mL) atroom temperature. After stirring for 24 hours at room temperature, thereaction solution was concentrated under reduced pressure. The residuewas purified by thin-layer chromatography (n-hexane/ethyl acetate=9/1)to give a mixture of trans-61 and cis-71 (99.5 mg, 190 μmol, 95.3%). Theratio of trans-61 and cis-71 was determined based on the ¹H NMRspectrum. The geometries of the respective compounds were confirmed byX-ray crystallographical analyses (CCDC 810934 (61) and CCDC 810841(71)).

1,8-Dihydro-1,8-bis(2-isopropylphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(61)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

Mp 298-300° C.;

R_(f)=0.70 (dichloromethane/methanol=24/1);

¹H NMR (500 MHz, CDCl₃) δ 0.76 (br s, 12H), 2.07 (br s, 2H), 6.95 (br,2H), 7.17 (dd, 2H, J=7.5, 7.5 Hz), 7.35-7.48 (m, 8H), 7.67 (br, 2H),7.77 (d, 2H, J=7.5 Hz);

¹³C NMR (126 MHz, CDCl₃) δ 21.9 (br, 2C), 24.2 (br, 2C), 27.8 (br, 2C),126.4 (4C), 126.8 (br, 2C), 128.4 (br, 2C), 128.5 (2C), 129.8 (4C),130.5 (br, 2C), 131.3 (2C), 132.3 (br, 2C), 133.7 (br, 2C), 135.8 (br,2C), 144.6 (2C), 145.4 (br, 2C);

IR (KBr, cm⁻¹) 611, 735, 766, 783, 908, 995, 1045, 1111, 1211, 1269,1360, 1454, 1493, 1512, 2967; HRMS (ESI⁺) m/z 523.2587 ([M+H]⁺, C3H₃₁N₆⁺ requires 523.2605);

C34H₃₀N₆: Calcd.: C, 78.13; H, 5.79; N, 16.08%; Found: C, 77.90; II,5.60; N, 15.85%.

1,10-Dihydro-1,10-bis(2-isopropylphenyl)dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis(triazole)(71)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

Mp>300° C.;

R_(f)=0.48 (dichloromethane/methanol=24/1);

¹H NMR (500 MHz, CDCl₃) δ 1.35 (br s, 6H), 1.45 (br s, 6H), 3.04 (br s,2H), 6.72-6.78 (m, 2H), 6.82-6.91 (m, 2H), 6.98-7.06 (m, 2H), 7.07-7.16(m, 2H), 7.40-7.49 (m, 2H), 7.52-7.61 (m, 4H), 7.79-7.87 (m, 2H);

¹³C NMR (126 MHz, CDCl₃) δ 23.1 (2C), 25.2 (2C), 28.6 (2C), 126.0 (2C),127.1 (2C), 127.5 (2C), 128.0 (2C), 129.1 (2C), 129.2 (2C), 130.4 (2C),130.7 (2C), 130.9 (2C), 131.0 (2C), 133.9 (2C), 134.5 (2C), 145.8 (2C),146.6 (2C);

IR (KBr, cm⁺¹) 523, 611, 733, 758, 910, 997, 1028, 1092, 1134, 1213,1261, 1360, 1452, 1495, 1512, 2965;

HRMS (ESI⁺) m/z 523.2585 ([M+H]⁺, C₃₄H₃₁N₆ ⁺ requires 523.2605).

Example 11 Production 11 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (14) above, to a solution of diyne 3(10.0 mg, 50 μmol) in methanol (5.00 mL) was added a solution of2,6-dimethylphenyl azide (4m) (17.7 mg, 120 μmol) in methanol (1.25 mL)at room temperature. After stirring for 30 minutes at room temperature,the reaction solution was concentrated under reduced pressure. Theresidue was purified by flash column chromatography (silica-gel 10 g,dichloromethane/methanol=9/1) to give a mixture of trans-6m and cis-7m(22.7 mg, 45.9 μmol, 91.9%). The ratio of trans-6m and cis-7m wasdetermined based on the ¹H NMR spectrum. The geometries of therespective compounds were confirmed by X-ray crystallographical analyses(CCDC 810935 (6m) and CCDC 810842 (7m)).

1,8-Bis(2,6-dimethylphenyl)-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(6m)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

Mp>300° C.;

R_(f)=0.49 (dichloromethane/methanol=49/1);

¹H NMR (500 MHz, CDCl₃) δ 1.53 (s, 6H), 2.41 (s, 6H), 6.87 (dd, 2H,J=1.0, 7.5 Hz), 6.94 (dd, 2H, J=5.0, 5.0 Hz), 7.19 (ddd, 2H, J=1.0, 7.5,7.5 Hz), 7.23 (d, 2H, J=5.0 Hz), 7.23 (d, 2H, J=5.0 Hz), 7.43 (ddd, 2H,J=1.0, 7.5, 7.5 Hz), 7.77 (dd, 211, J=1.0, 7.5 Hz);

¹³C NMR (126 MHz, CDCl₃) δ 17.3 (2C), 18.4 (2C), 126.4 (2C), 128.4 (2C),128.5 (2C), 128.6 (2C), 129.4 (2C), 129.9 (2C), 130.0 (2C), 131.4 (2C),132.0 (2C), 134.3 (2C), 135.7 (2C), 135.8 (2C), 136.1 (2C), 144.7 (2C);

IR (KBr, cm⁻¹) 735, 764, 781, 912, 995, 1113, 1354, 1427, 1474, 1512;

HRMS (ESI⁺) m/z 495.2276 ([M+H]⁺, C₃₂H₂₇N₆ ⁺ requires 495.2292);

C₃₂H₂₆N₆: Calcd.: C, 77.71; H, 5.30; N, 16.99%; Found: C, 77.95; H,5.41; N, 16.88%.

1,10-Bis(2,6-dimethylphenyl)-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazo le) (7m)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

Mp>300° C.;

R_(f)=0.24 (dichloromethane/methanol=49/1);

¹H NMR (500 MHz, CDCl₃) δ 1.76 (s, 6H), 2.23 (s, 6H), 6.87-6.93 (m, 2H),7.00 (d, 2H, J=7.5 Hz), 7.03-7.08 (m, 2H), 7.22-7.29 (m, 4H), 7.53-7.58(m, 2H), 7.74-7.79 (m, 2H);

¹³C NMR (126 MHz, CDCl₃) δ 18.4 (2C), 18.8 (2C), 127.0 (2C), 128.2 (2C),128.8 (2C), 129.1 (2C), 129.3 (2C), 130.2 (2C), 130.6 (2C), 130.7 (2C),131.1 (2C), 134.2 (2C), 134.8 (2C), 135.3 (2C), 137.3 (2C), 146.1 (2C);

IR (KBr, cm⁻¹) 704, 733, 764, 918, 995, 1134, 1267, 1290, 1346, 1474,1508;

HRMS (ESI⁺) m/z 495.2315 ([M+H]⁺, C₃₂H₂₇N₆ ⁺ requires 495.2292);

C₃₂H₂₆N₆: Calcd.: C, 77.71; H, 5.30; N, 16.99%; Found: C, 77.59; H,5.33; N, 16.73%.

Example 12 Production 12 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (15) above, to a solution of diyne 3(10.0 mg, 50 μmol) in methanol (5.00 mL) was added a solution of2,6-dietylphenyl azide (4n) (21.0 mg, 120 μmol) in methanol (1.25 mL) atroom temperature. After stirring for 30 minutes at room temperature, thereaction solution was concentrated under reduced pressure. The residuewas purified by flash column chromatography (silica-gel 10 g,dichloromethane only to dichloromethane/methanol=24/1) to give a mixtureof trans-6n and cis-7n (25.7 mg, 46.7 mmol, 93.4%). The ratio oftrans-6n and cis-7n was determined based on the ¹H NMR spectrum. Thegeometries of the respective compounds were confirmed by X-raycrystallographical analyses (CCDC 810936 (6n) and CCDC 810843 (7n)).

1,8-Bis(2,6-diethylphenyl)-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(6n)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

Mp 255-257° C.;

R_(f)=0.57 (dichloromethane/methanol=49/1);

¹H NMR (500 MHz, CDCl₃) δ 0.73 (dd, 6H, J=7.5, 7.5 Hz), 1.41 (dd, 6H,J=7.5, 7.5 Hz), 1.74 (dq, 2H, J=7.5, 15.0 Hz), 1.92 (dq, 2H, J=7.5, 15.0Hz), 2.59 (dq, 2H, J=7.0, 14.0 Hz), 2.64 (dq, 2H, J=7.0, 14.0 Hz), 6.85(dd, 2H, J=1.0, 7.5 Hz), 7.00 (d, 2H, J=7.5 Hz), 7.17 (ddd, 2H, J=1.0,7.5, 7.5 Hz), 7.30 (d, 2H, J=7.5 Hz), 7.35 (dd, 2H, J=7.5, 7.5 Hz), 7.41(ddd, 2H, J=1.0, 7.5, 7.5 Hz) 7.76 (dd, 2H, J=1.0, 7.5 Hz);

¹³C NMR (126 MHz, CDCl₃) δ 13.5 (2C), 15.2 (2C), 24.0 (2C), 24.7 (2C),126.45 (2C), 126.49 (2C), 126.57 (2C), 128.5 (2C), 129.4 (2C), 129.9(2C), 130.3 (2C), 131.4 (2C), 132.2 (2C), 133.15 (2C), 135.9 (2C), 141.0(2C), 142.1 (2C), 144.6 (2C);

IR (KBr, cm⁻¹) 733, 758, 910, 995, 1358, 1466, 1510, 2876, 2936, 2970;

HRMS (ESI⁺) m/z 551.2912 ([M+H]⁺, C₃₆H₃₅N₆ ⁺ requires 551.2918);

C₃₆H₃₄N₆: Calcd.: C, 78.52; H, 6.22; N, 15.26%; Found: C, 78.26; H,6.47; N, 14.97%.

1,10-Bis(2,6-diethylphenyl)-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis(triazole) (7n)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

Mp>300° C.;

R_(f)=0.30 (dichloromethane/methanol=49/1);

¹H NMR (500 MHz, CDCl₃) δ 0.91 (dd, 6H, J=7.5, 7.5 Hz), 1.32 (dd, 6H,J=7.5, 7.5 Hz), 2.04 (dq, 2H, J=7.5, 15.0 Hz), 2.09 (dq, 2H, J=7.5, 15.0Hz), 2.26 (dq, 2H, J=7.5, 15.0 Hz), 2.41 (dq, 2H, J=7.5, 15.0 Hz),6.83-6.87 (m, 2H), 6.96-7.01 (m, 2H), 7.08 (d, 2H, J=7.5 Hz), 7.31 (d,2H, J=7.5 Hz), 7.40 (dd, 2H, J=7.5, 7.5 Hz), 7.53-7.57 (m, 2H),7.74-7.79 (m, 2H);

¹³C NMR (126 MHz, CDCl₃) δ 14.9 (2C), 15.2 (2C), 24.7 (2C), 25.3 (2C),126.7 (2C), 126.8 (2C), 126.9 (2C), 129.0 (2C), 129.1 (2C), 130.6 (2C),130.7 (2C), 130.8 (2C), 131.2 (2C), 133.6 (2C), 134.2 (2C), 140.9 (2C),143.3 (2C), 145.9 (2C);

IR (KBr, cm⁻¹) 733, 758, 783, 908, 993, 1061, 1113, 1128, 1261, 1352,1470, 1508, 2872, 2932, 2965;

HRMS (ESI⁺) m/z 551.2940 ([M+H]⁺, C₃₆H₃₅N₆ ⁺ requires 551.2918);

C₃₆H₃₄N₆: Calcd.: C, 78.52; H, 6.22; N, 15.26%; Found: C, 78.49; H,6.20; N, 15.03%.

Example 13 Production 13 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (16) above, to a solution of diyne 3(20.3 mg, 102 μmol) in methanol (9.0 mL) was added a solution of2-t-butyl-6-methylphenyl azide (4o) (45.4 mg, 240 μmol) in methanol (3.5mL) at room temperature. After stirring for 24 hours at roomtemperature, the reaction solution was concentrated under reducedpressure. The residue was purified by flash column chromatography(silica-gel 10 g, hexane only to ethyl acetate) to give a mixture ofdiastereomers of trans-6o and cis-7o (58.4 mg, 101 μmol, 99.7%).

1,8-Bis(2-tert-butyl-6-methylphenyl)-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(6o, a mixture of diastereomers)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

R_(f)=0.47 (dichloromethane/methanol=3/1);

¹H NMR (400 MHz, CDCl₃) (major isomer) δ 0.87 (s, 18H), 2.07 (s, 6H),6.77 (dd, 2H, J=1.2, 8.0 Hz), 7.13 (ddd, 2H, J=1.2, 7.6, 7.6 Hz),7.23-7.26 (m, 2H), 7.34-7.36 (m, 4H), 7.39 (dd, 2H, J=1.2, 8.0 Hz), 7.69(dd, 2H, J=1.2, 7.6 Hz).

1,10-Bis(2-tert-butyl-6-methylphenyl)-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(7o, a mixture of diastereomers)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

R_(f)=0.30 (dichloromethane/methanol=3/1);

¹H NMR (400 MHz, CDCl₃) (major isomer) δ 0.93 (s, 18H), 1.80 (s, 6H),6.98-7.02 (m, 2H), 7.30-7.42 (m, 6H), 7.51-7.57 (m, 4H), 7.63-7.67 (m,2H).

Example 14 Production 14 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (17) above, to a solution of diyne 3(20.0 mg, 100 μmol) in methanol (12.5 mL) was added 2,6-dibromophenylazide (4p) (66.5 mg, 240 μmol) at room temperature. After stirring for 9hours at room temperature, the reaction solution was concentrated underreduced pressure. The residue was purified by thin-layer chromatography(dichloromethane) to give a mixture of trans-6p and cis-7p (74.3 mg,98.7 μmol, 98.7%). The ratio of trans-6p and cis-7p was determined basedon the ¹H NMR spectrum.

1,8-Bis(2,6-dibromophenyl)-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(6p)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

R_(f)=0.07 (dichloromethane);

¹H NMR (400 MHz, CDCl₃) δ 7.16-7.27 (m, 6H), 7.46-7.48 (m, 4H), 7.75(dd, 2H, J=1.2, 8.4 Hz), 782 (dd, 2H, J=0.8, 7.6 Hz).

1,10-Bis(2,6-dibromolphenyl)-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(7p)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

R_(f)=0.01 (dichloromethane);

¹H NMR (400 MHz, CDCl₃) δ 7.16 (dd, 2H, J=3.6, 6.0 Hz), 7.22-7.30 (m,4H), 7.53-7.57 (m, 4H), 7.75-7.79 (m, 4H).

Example 15 Production 15 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (18) above, to a solution of diyne 3(20.4 mg, 102 μmol) in methanol (9.0 mL) was added a solution of3,5-bis(trifluoromethyl)phenyl azide (4q) (61.2 mg, 240 μmol) inmethanol (3.5 mL) at room temperature. After stirring for 20 hours atroom temperature, the reaction solution was concentrated under reducedpressure. The residue was purified by thin-layer chromatography(dichloromethane/hexane=3/1) to give a mixture of trans-6q and cis-7q(71.6 mg, 101 μmol, 99.0%). The ratio of trans-6q and cis-7q wasdetermined based on the ¹H NMR spectrum.

1,8-Bis[3,5-bis(trifluoromethyl)phenyl]-1,8-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(6q)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

¹H NMR (400 MHz, CDCl₃) δ 6.84 (dd, 2H, J=0.8, 8.0 Hz), 7.32 (ddd, 2H,J=1.2, 7.6, 7.6 Hz), 7.60 (ddd, 2H, J=1.2, 7.6, 7.6 Hz), 7.75 (s, 4H),7.90-7.93 (m, 4H).

1,10-Bis[3,5-bis(trifluoromethyl)phenyl]-1,10-dihydrodibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole) (7q)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

¹H NMR (400 MHz, CDCl₃) δ 7.00-7.03 (m, 2H), 7.34-7.38 (m, 2H),7.58-7.61 (m, 2H), 7.75-7.78 (m, 2H), 7.88 (d, 4H, J=1.6 Hz), 7.99 (s,2H).

Example 16 Production 16 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (19) above, to a solution of diyne 3(40.0 mg, 200 μmol) in methanol (23.5 mL) was added a solution of4-(azidomethyl)benzyl alcohol (4d, synthesized by the method describedin Non-Patent Document M. Smet, K. Metten, W. Dehaen, Collect. Czech.Chem. Commun. 2004, 69, 1097-1108) (78.3 mg, 480 p,mol) in methanol (1.5mL) at room temperature. After stirring for 60 minutes at the sametemperature, the reaction solution was concentrated under reducedpressure using an evaporator. The residue was purified by flash columnchromatography (silica-gel 10 g, dichloromethane/methanol=29/1) to givetwo regioisomeric bis-cycloadducts of trans-6d (63.5 mg, 121 μmol,60.3%) and cis-7d (40.9 mg, 77.7 wnol, 38.9%). The geometries of therespective compounds were confirmed by X-ray crystallographical analyses(CCDC 759899 (6d) and CCDC 759904 (7d)).

1,8-Dihydro-1,8-bis[4-(hydroxylmethyl)benzyl]dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(6d)

Recrystallization from dichloromethane/methanol gave colorless crystals.The following physical properties of the crystals were measured toconduct structural analysis.

Mp>300° C. (dec.);

R_(f)=0.45 (hexane/ethyl acetate=1/9);

¹H NMR (300 MHz, CDCl₃) δ 3.38 (br s, 2H), 4.52-4.70 (m, 4H), 5.27 (d,2H, J=14.7 Hz), 5.29 (d, 2H, J=14.7 Hz), 6.52-6.62 (AA′BB′ x2, 4H),7.06-7.13 (AA′BB′ x2, 4H), 7.13-7.20 (m, 2H), 7.38-7.57 (m, 6H);

¹³C NMR (75.5 MHz, CDCl₃) δ 51.0 (2C), 62.4 (2C), 126.2 (4C), 126.5(4C), 129.3 (2C), 130.1 (2C), 130.3 (2C), 130.9 (2C), 132.1 (2C), 134.0(2C), 134.5 (2C), 142.1 (2C), 144.5 (2C), 147.7 (2C);

IR (KBr, cm⁻¹) 519, 594, 775, 986, 1028, 1105, 1132, 1215, 1252, 1314,1352, 1422, 1474, 1514, 1616, 2097, 2868, 3366;

HRMS (ESI⁺) m/z 527.2191 ([M+H]⁺, C₃₂H₂₇N₆O₂ ⁺ Calcd. 527.2190).

1,10-Dihydro-1,10-bis[4-(hydroxylmethyl)benzyl]dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(7d)

Recrystallization from dichloromethane/methanol gave colorless crystals.The following physical properties of the crystals were measured toconduct structural analysis.

Mp>300° C. (dec.);

R_(f)=0.39 (hexane/ethyl acetate=1/9);

¹H NMR (300 MHz, CDCl₃) δ 2.03 (t, 2H, J=5.2 Hz), 4.66 (d, 4H, J=5.2Hz), 4.96 (d, 2H, J=15.7 Hz), 5.33 (d, 2H, J=15.7 Hz), 6.90-7.00 (AA′BB′x2, 4H), 7.08-7.16 (m, 2H) 7.20-7.32 (AA′BB′ x2, 4H), 7.40-7.52 (m, 4H),7.62-7.70 (m, 2H);

¹³C NMR (75.5 MHz, CDCl₃) δ 51.3 (2C), 62.5 (2C), 126.8 (4C), 127.0(2C), 127.1 (4C), 129.2 (2C), 130.2 (2C), 130.3 (2C), 130.7 (2C), 130.8(2C), 133.5 (2C), 133.8 (2C), 142.5 (2C), 145.0 (2C);

IR (KBr, cm⁻¹), 704, 737, 764, 986, 1028, 1134, 1209, 1248, 1265, 1287,1315, 1346, 1422, 1514, 1634, 2089, 3360;

HRMS (ESI⁺) m/z 527.2205 ([M+H]⁻¹, C₃₂H₂₇N₆O₂ ⁺ Calcd. 527.2190).

Example 17 Production 17 of Bis-Cycloadducts by SPDC Reaction

As shown in the reaction scheme (20) above, to a solution of diyne 3(40.0 mg, 200 μmol) in methanol (23.5 mL) was added a solution of methyl4-(azidomethyl)benzoate (4e, synthesized by the method described inNon-Patent Document E. A. Wydysh, S. M. Medghalchi, A. Vadlamudi, and C.A. Townsend, J. Med. Chem. 2009, 52, 3317-3327) (91.8 mg, 480 μmol) inmethanol (1.5 mL) at room temperature. After stirring for 120 minutes atthe same temperature, the reaction solution was concentrated underreduced pressure using an evaporator. The residual solid was placed on aKiriyama funnel and washed with ethyl acetate to give purecis-bis-cycloadduct 7e (36.1 mg, 61.9 μmol, 30.9%) as one of the twobis-cycloadduct regioisomers. The filtrate was concentrated underreduced pressure using an evaporator and the residue was purified byflash column chromatography (silica-gel 10 g, hexane/ethyl acetate=1/2to ethyl acetate only) to give trans-bis-cycloadduct 6e (71.8 mg, 123μmol, 61.5%). The geometries of these compounds were confirmed by theirreduction of the esters to the corresponding diols 6d and 7d,respectively, using LiAlH₄ in THF (0° C. to heating under reflux, 7.5hours).

1,8-Dihydro-1,8-bis[4-(methoxycarbonyl)benzyl]dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(6e)

The following physical properties of the colorless solid obtained weremeasured to conduct structural analysis.

Mp 102-104° C.;

R_(f)=0.31 (hexane/ethyl acetate=2/1);

R_(f)=0.27 (dichloromethane/methanol=15/1);

¹H NMR (300 MHz, CDCl₃) δ 3.90 (s, 6H), 5.34 (d, 2H, J=15.9 Hz), 5.57(d, 2H, J=15.9 Hz), 7.03 (d, 2H, J=7.6 Hz), 7.05-7.12 (AA′BB′ x2, 4H),7.39 (dd, 2H, J=7.6, 7.6 Hz), 7.54 (dd, 2H, J=7.6, 7.6 Hz), 7.73 (d, 2H,J=7.6 Hz), 7.91-8.00 (AA′BB′ x2, 4H);

¹³C NMR (75.5 MHz, CDCl₃) δ 51.0 (2C), 52.1 (2C), 126.0 (2C), 126.5(4C), 128.9 (2C), 129.3 (4C), 129.4 (2C), 130.1 (2C), 130.4 (2C), 130.7(2C), 131.8 (2C), 134.6 (2C), 140.9 (2C), 144.8 (2C), 165.6 (2C);

IR (KBr, cm⁻¹) 581, 735, 746, 764, 806, 910, 1020, 1111, 1180, 1283,1352, 1435, 1516, 1614, 1719, 2951;

HRMS (ESI⁺) m/z 583.2104 ([M+H]⁺, C₃₄H₂₇N₆O₄ ⁺ Calcd. 583.2088).

1,10-Dihydro-1,10-bis[4-(methoxycarbonyl)benzyl]dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d]bis([1,2,3]triazole)(7e)

The following physical properties of the colorless solid obtained weremeasured to conduct structural analysis.

Mp 287-290° C.;

R_(f)=0.31 (hexane/ethyl acetate=2/1);

R_(f)=0.27 (dichloromethane/methanol=15/1);

¹H NMR (300 MHz, CDCl₃) δ 3.91 (s, 6H), 4.98 (d, 2H, J=15.9 Hz), 5.36(d, 2H, J=15.9 Hz), 6.98-7.11 (m, 6H), 7.38-7.45 (m, 2H), 7.46-7.56 (m,2H), 7.64-7.72 (m, 2H), 7.94-8.00 (AA′BB′ x2, 411);

¹³C NMR (75.5 MHz, CDCl₃) δ 51.7 (2C), 52.4 (2C), 127.1 (4C), 127.8(2C), 129.3 (2C), 130.06 (2C), 130.14 (2C), 130.2 (4C), 130.3 (2C),130.4 (2C), 130.6 (2C), 133.5 (2C), 140.1 (2C), 146.3 (2C), 166.3 (2C);

IR (KBr, cm⁻¹) 582, 735, 750, 764, 804, 910, 1020, 1111, 1180, 1283,1435, 1516, 1614, 1719, 2951;

HRMS (ESI⁺) m/z 583.2092 ([M+H]⁺, C₃₄H₂₇N₆O₄ ⁺ Calcd. 583.2088).

Example 18 Production 18 of Bis-Cycloadducts by SPDC Reaction

As illustrated in the reaction scheme (21), to a solution of diyne 3(40.0 mg, 200 μmol) in methanol (22 mL) was added a mixture of4-(azidomethyl)benzyl alcohol (4d) (39.2 mg, 240 μmol) and methyl4-(azidomethyl)benzoate (4e) (45.9 mg, 240 μmol) in methanol (3 mL) atroom temperature. After stirring for 120 minutes at the sametemperature, the reaction solution was concentrated under reducedpressure using an evaporator. The residue was then purified by flashcolumn chromatography (silica-gel 10 g, hexane/ethyl acetate=1/1 to 1/4to ethyl acetate only) to give two regioisomeric hetero or unsymmetricalbis-cycloadducts of trans-6de (21.8 mg, 39.3 μmol, 19.7%) and cis-7de(25.3 mg, 45.6 μmol, 22.8%). In addition, two pairs of the homo orsymmetrical bis-cycloadducts of trans-6d/cis-7d (29.2 mg, 55.5 μmol,27.8%, 6d/7d=1.8/1) and trans-6e/cis-7e (31.4 mg, 53.9 μmol, 27.0%,6e/7e=1.2/1) were obtained as a mixture of regioisomers, respectively.The isomeric ratios in the regioisomers were determined by comparing theintegration ratios of benzylic proton peaks on ¹H NMR spectrum. Thegeometries of 6de and 7de were confirmed by reduction of the esters tothe corresponding diols 6d and 7d, respectively, through reaction withlithium aluminum hydride (LiAlH₄) in tetrahydrofuran (THF) (0° C. toheating under reflux, 7.5 hours).

1,8-Dihydro-1-[4-(hydroxylmethyl)benzyl]-8-[4-(methoxycarbonyl)benzyl]dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(6de)

The following physical properties of the colorless solid obtained weredetermined to perform structural analysis.

Mp 116-118° C.;

R_(f)=0.30 (hexane/ethyl acetate=1/4);

¹H NMR (300 MHz, CDCl₃) δ 3.16 (br s, 1H), 3.91 (s, 3H), 4.60 (s, 2H),5.28-5.40 (m, 2H), 5.51 (d, 1H, J=15.5 Hz), 5.61 (d, 1H, J=15.5 Hz),6.64-6.72 (AA′BB′, 2H), 6.92-7.03 (m, 3H), 7.05-7.12 (AA′BB′, 2H), 7.23(d, 1H, J=7.3 Hz), 7.38 (dd, 1H, J=7.3, 7.3 Hz), 7.42-7.62 (m, 4H), 7.71(d, 1H, J=7.3 Hz), 7.90-7.98 (AA′BB′, 2H);

¹³C NMR (75.5 MHz, CDCl₃) δ 51.6, 52.1, 52.6, 64.8, 126.0, 126.5, 126.6(2C), 127.2 (2C), 127.8 (2C), 128.8, 128.9, 129.67, 129.71, 130.0, 130.1(3C), 130.4, 131.0, 131.2, 131.9, 132.5, 133.7, 134.7, 134.9, 139.9,141.3, 144.6, 145.2, 166.4;

IR (KBr, cm⁻¹) 583, 748, 961, 986, 1018, 1049, 1109, 1182, 1215, 1281,1352, 1435, 1514, 1614, 1719, 2068, 2359, 3389;

HRMS (ESI⁺) m/z 555.2150 ([M+H]⁺, C₃₃H₂₇N₆O₃ ⁺ Calcd. 555.2139).

1,10-Dihydro-1-[4-(hydroxylmethyl)benzyl]-10-[4-(methoxycarbonyl)benzyl]dibenzo[3,4:7,8]cycloocta[1,2-d:5,6-d′]bis([1,2,3]triazole)(7de)

The following physical properties of the colorless crystals obtainedwere determined to perform structural analysis.

Mp 134-136° C.;

R_(f)=0.38 (hexane/ethyl acetate=1/4);

¹H NMR (300 MHz, CDCl₃) δ 1.90 (t, 1H, J=5.4 Hz), 3.90 (s, 3H), 4.68 (d,2H, J=5.4 Hz), 4.89 (d, 1H, J=16.0 Hz), 5.04 (d, 1H, J=16.0 Hz), 5.33(d, 1H, J=16.0 Hz), 5.39 (d, 1H, J=16.0 Hz), 6.88-6.96 (AA′BB′, 2H),7.00 (d, 1H, J=7.6 Hz), 7.04-7.10 (AA′BB′, 2H), 7.15 (d, 1H, J=7.6 Hz),7.24-7.32 (AA′BB′, 2H), 7.34-7.54 (m, 4H), 7.62-7.70 (m, 2H), 7.92-7.99(AA′BB′, 2H);

¹³C NMR (75.5 MHz, CDCl₃) δ 51.6, 51.9, 52.3, 64.3, 127.0 (2C), 127.1(2C), 127.4 (2C), 127.6, 128.0, 129.18, 129.22, 129.9, 130.06, 130.09(3C), 130.3, 130.5, 130.61, 130.64, 130.7, 133.4, 133.7, 134.1, 140.4,141.3, 146.1, 146.2, 150.8, 166.4;

IR (KBr, cm⁻¹) 471, 579, 750, 984, 1018, 1111, 1209, 1281, 1346, 1416,1514, 1614, 1715, 2949, 3061, 3416;

HRMS (ER⁺) m/z 555.2127 ([M+H]⁺, C₃₃H₂₇N₆O₃ ⁺ Calcd. 555.2139).

As illustrated in the SPDC reaction of EXAMPLE 18 above, the hetero orunsymmetrical bis-cycloadduct can be obtained as the main product byusing the two different azide compounds.

As shown in the results of EXAMPLES above, it is revealed that variousazide compounds are applicable to the SPDC reaction. The substituent Rin the vicinity of the azido group in azide compounds is modified tovarious types, whereby the substituent on the triazole ring in thebis-cycloadducts produced can be modified appropriately as shown informula (1) or (2) described above.

As in the EXAMPLE above, the SPDC reaction proceeds rapidly even undercatalyst-free, mild conditions at not very high temperatures. This isconsidered because the reaction with azide compounds would be promotedby strain of the cyclic diyne compound containing an 8-membered cyclicskeleton and proceed spontaneously. Furthermore, the SPDC reaction isefficient since two azide compounds can be added and ligated to a cyclicdiyne in a single step.

On the other hand, when, e.g., an azido group and a triarylphosphinederivative are used to modify a biomolecule or in the case of a singleclick reaction wherein a single azide compound is added to an alkyne, aplurality of steps are required. This is because it becomes necessary toproduce a functional azide compound including fluorescence labeling as aprobe and react to add the functional azide compound to a biomoleculeeach time depending upon the purpose of experimentation.

In the SPDC reaction of diyne 3 with methyl azide 4f shown by thereaction scheme (22) above, activation energy and transition state werecalculated using a density functional theory: DFT [B3LYP/6-31G(d)]. Asshown in FIG. 1, the results indicate that energy barriers forcycloaddition of methyl azide 4f to diyne 3 are low and methyl azide 4fis spontaneously reactive with both diyne 3 and monoyne intermediate 5fat room temperature. In particular, the activation energies for a secondcycloaddition reaction of monoyne intermediate 5f (+8.8 and +9.5kcal/mol for trans- and cis-additions, respectively) were smaller thanthe activation energy for a first cycloaddition reaction of diyne 3(+12.4 kcal/mol). This indicates that monoyne intermediate 5f has ahigher reactivity with methyl azide 4f than diyne 3 does. Therefore,when an addition reaction of two azide compounds proceeds stepwise inthe SPDC reaction, a second azido group can also be introduced promptly.

Example 19 Comparison in Reactivity Between SPDC Reaction andConventional Single Click Reaction

To compare the SPDC reaction with a conventional strain-promoted singleclick reaction, the competition experiment was performed. Morespecifically, to a mixture of diyne 3 (20.0 mg, 100 μmol) and monoyne 2a(11,12-didehydro-5,6-dihydrodibenzo[a,e]cycloocten-5-ol synthesized bythe method described in Non-Patent Document X. Ning, J. Guo. M. A.Wolfert, G.-J. Boons, Angew. Chem. Int. Ed. 2008, 47, 2253-2255) (22.0mg, 100 pt,mol) dissolved in methanol (22.5 mL) was added a solution ofbenzyl azide (4a) (13.3 mg, 100 p.inol) in methanol (2.5 mL) at roomtemperature. After stirring for 24 hours at the same temperature, thereaction solution was concentrated under reduced pressure using anevaporator. The residue was a mixture of two bis-cycloadducts, trans6a/cis 7a from diyne 3 and mono-cycloadducts 23-26 from monoyne 2a; itwas difficult to separate chromatographically or determine the ratio ofbis- and mono-cycloadducts by ¹H NMR as they were. Thus, an oxidant wasadded to the mixture to convert the mono-cycloadduct alcohols 23-26 frommonoyne 2a into the corresponding ketones 27 (trans) and 28 (cis) in thereaction scheme (24) described below to make their analysis easy.Specifically, to a solution of the above residue in chloroform (10 mL)was added Dess-Martin periodinane (37.2 mg, 87.7 μmol) at roomtemperature. After stirring for 4 hours at the same temperature, thereaction solution was concentrated under reduced pressure using anevaporator. The residue was passed through a short silica-gel column (5g, ethyl acetate only) to give a mixture of bis-cycloadducts,trans-6a/cis-7a and mono-cycloadduct ketones, trans-27/cis-28. Bycomparing the integration values of protons on ¹H NMR spectrum of themixture, the ratio of 6a/7a to 27/28 was determined to be approximately1:1. This indicates the similar reactivity of diyne 3 and monoyne 2atoward benzyl azide (4a).

Authentic samples of the ketones 27 (trans) and 28 (cis) produced inEXAMPLE 19 above were synthesized as shown by the reaction scheme (24)above. More specifically, to a solution of monoyne 2a (108 mg, 490 μmol)in methanol (11 mL) was added a solution of benzyl azide (4a) (163 mg,1.22 mmol) in methanol (4 mL) at room temperature. After stirring for 2hours at the same temperature, the reaction solution was concentratedunder reduced pressure using an evaporator. The residue was thenpurified by flash column chromatography (silica-gel 10 g,dichloromethane/methanol=150/1 to 9/1) to give a mixture oftrans-alcohols 23/24 (R_(f)=0.14 (dichloromethane/methanol=49/1)) (72.8mg, 206 μmol, 42.0% based on the azide (4a)) and a mixture ofcis-alcohols 25/26 (R_(f)=0.22 (dichloromethane/methanol=49/1)) (83.0mg, 235 μmol, 47.9% based on the azide (4a)), among the fourregioisomers in the mono-cycloadducts.

To a solution of the mixture of trans-alcohols 23/24 (25.8 mg, 73.0μmol) in dichloromethane (10 mL) was added Dess-Martin periodinane (37.2mg, 87.7 μmol) at room temperature. After stirring for 21 hours at thesame temperature, the reaction solution was concentrated under reducedpressure using an evaporator. The residue was purified by successiveflash column chromatography (silica-gel 10 g, ethyl acetate only, andthen silica-gel 10 g, dichloromethane only) to give trans-ketone 27(23.3 mg, 66.3 μmol, 90.8%).

On the other hand, to a solution of the mixture of cis-alcohols 25/26(22.3 mg, 63.1 μmol) in dichloromethane (10 mL) was likewise addedDess-Martin periodinane (32.1 mg, 75.7 μmol) at room temperature. Afterstirring for 21 hours at the same temperature, the reaction solution wasconcentrated under reduced pressure using an evaporator. The residue waspurified by successive flash column chromatography (silica-gel 10 g,ethyl acetate only, and then silica-gel 10 g, dichloromethane only) togive cis-ketone 28 (20.5 mg, 58.3 μmol, 92.5%).

The geometries of the respective ketones were confirmed by X-raycrystallographical analyses (CCDC 761157 (27) and CCDC 761156 (28)).

1-Benzyl-1H-dibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazol-8(9H)-one(27)

Recrystallization from n-hexane/ethyl acetate gave colorless crystals.The following physical properties of the crystals were measured toconduct structural analysis.

Mp 220-222° C.;

R_(f)=0.55 (dichloromethane/methanol=9/1);

¹H NMR (300 MHz, CDCl₃) δ 3.66 (d, 1H, J=12.1 Hz), 3.76 (d, 1H, J=12.1Hz), 5.57 (d, 1H, J=15.1 Hz), 5.69 (d, 1H, J=15.1 Hz), 7.03-7.10 (m,2H), 7.23-7.37 (m, 5H), 7.38-7.52 (m, 3H), 7.64 (ddd, 1H, J=1.4, 8.0,8.0 Hz), 8.01 (dd, 1H, J=1.4, 8.0 Hz), 8.29 (dd, 1H, J=1.4, 8.0 Hz);

¹³C NMR (75.5 MHz, CDCl₃) δ 48.0, 52.5, 125.4, 127.2 (2C), 127.5, 128.2,128.29, 128.34 (3C), 128.8, 129.8, 130.8, 130.9, 131.3, 133.0, 133.2,133.8, 134.0, 135.0, 146.6, 195.4;

IR (KBr, cm⁻¹) 542, 603, 704, 735, 764, 908, 1013, 1150, 1207, 1256,1279, 1346, 1431, 1454, 1497, 1597, 1668, 3063

1-Benzyl-1H-dibenzo[3,4:7,8]cycloocta[1,2-d][1,2,3]triazol-9(8H)-one(28)

Recrystallization from n-hexane/ethyl acetate gave colorless crystals.The following physical properties of the crystals were measured toconduct structural analysis.

Mp 178-180° C.;

R_(f)=0.67 (dichloromethane/methanol=9/1);

¹H NMR (300 MHz, CDCl₃) δ 3.60 (br, 1H), 3.81 (br, 1H), 5.67 (d, 2H,J=5.5 Hz), 7.01-7.09 (m, 2H), 7.18 (dd, 1H, J=1.7, 7.7 Hz), 7.24-7.30(m, 3H), 7.32-7.41 (m, 3H), 7.43-7.56 (m, 2H), 7.66-7.74 (m, 1H), 8.15(dd, 1H, J=1.7, 7.7 Hz);

¹³C NMR (75.5 MHz, CDCl₃) δ 49.4, 53.1, 126.3, 127.3 (2C), 127.8, 128.5,128.7, 128.9 (2C), 129.4, 129.5, 129.6, 130.5, 131.2, 131.4, 132.4,133.2, 134.5, 134.8, 136.1, 145.8, 196.9;

IR (KBr, cm⁻¹) 538, 604, 706, 730, 766, 908, 1028, 1157, 1211, 1250,1281, 1352, 1429, 1454, 1497, 1597, 1672, 3063

Example 20 Kinetic Study for SPDC Reaction

To compare the difference in reaction rate between the SPDC reaction andthe single click reaction more quantitatively, the kinetic study for theSPDC reaction shown by the reaction scheme (25) above was carried out.That is, second order rate constants of the first cycloaddition in theSPDC reaction were monitored by reacting an excess amount of benzylazide (4a) with diyne 3 and measuring time dependent decreases of thediyne 3.

More specifically, to 1.5 mL of 2.0 mM (final concentration at 1.0 mM)solution of diyne 3 in methanol placed in a quartz cuvette of a 10 mmlight path, was added 1.5 mL of methanol solution of benzyl azide (4a)in three different concentrations (20, 100 or 200 mM; finalconcentration at 10, 50 and 100 mM, respectively), while keepingtemperature at 25° C. Immediately after initiation of the reaction, theabsorbance focusing at 351.5 nm (loge=3.22), which is an absorptionwavelength characteristic of diyne 3, was monitored by UV spectroscopy.For information, almost no significant absorption for benzyl azide (4a)and bis-cycloadducts 6a/7a was observed at 351.5 nm. The monitoringabove was continued for 300-3000 seconds and the above measurements wererepeated in triplicate for each concentration of benzyl azide (4a)described above. By plotting the absorbance data versus time (second),the exponential decay curves were obtained as shown in FIG. 2. Theapproximation curves were determined by least-squares fitting usingKaleidaGraph (vera 4.1.1.) to calculate the pseudo-first order rateconstants (k″). As such, the pseudo-first order rate constants (k″) wereplotted versus each concentration of benzyl azide (4a) to give astraight line shown in FIG. 3. A linear regression analysis usingMicrosoft Office Excel 2007 was conducted and as a result, the slope ofthis straight line, namely, the second order rate constant for the firstcycloaddition in the SPDC reaction, which is the rate-determining stepof the SPDC reaction, was found to be (6.29±0.05 SE)×10⁻² M⁻¹s⁻¹.

The second order rate constant of monoyne 2a with benzyl azide (4a) inthe single click reaction under the same conditions (25° C. in methanol)is reportedly (5.67±0.27 SE)×10⁻² M⁻¹s⁻¹ (25° C. in methanol)(Non-Patent Document: A. A. Poloukhtine, N. E. Mbua, M. A. Wolfert,G.-J. Boons, V. V. Popik, J. Am. Chem. Soc. 2009, 131, 15769-15776). Itwas thus shown that both reactions proceed at almost all the same ratesalso from a kinetic viewpoint.

Example 21 Chemical Modification of Azido-Biomolecules)

Next, EXAMPLE 21 is described for chemical modification ofazido-biomolecules by the SPDC reaction. In EXAMPLE 21, expressionvector pGEX6P-1-HaloTag capable of expressing the fusion proteinGST-HaloTag protein between GST protein and HaloTag protein was firstprepared. The expression vector was then transformed into Escherichiacoli. Expression was induced by the addition of IPTG (isopropylthiogalactoside) to give the GST-HaloTag protein. The HaloTag-GSTprotein was purified from the E. coli lysate using a GSH-Sepharose resinon which GST is specifically bound. The GST-HaloTag protein thuspurified was used for the following chemical modification in the stateimmobilized onto the GSH-Sepharose resin (hereinafter referred to asHaloTag-Sepharose resin). This HaloTag-Sepharose resin was reacted withHaloTag ligand possessing a long-chain chloroalkane and an azido group(azido-HaloTag ligand) 8 (see formula (26) below) to give the desiredazido-HaloTag-Sepharose resin. The fluorescence modification ofazido-HaloTag-Sepharose resin was attempted in the SPDC reaction byadding diyne 3 to a solution of the azido-HaloTag-Sepharose resin thusobtained and TESRA-PEO₃-azide (9) (an azido-conjugatedtetraethylsulforhodamine (TESRA) derivative, cf., formula (26)). Afterthe reaction described above, to the azido-HaloTag-Sepharose resin wasfirst added a SDS-sample buffer containing a reducing agent. Theligation between GST and GSH was cleaved by heating, and the modifiedGST-HaloTag protein was excised from the resin. These proteins wereseparated using an acrylamide gel (SDS-PAGE), and then analyzed in afluorescence imaging analyzer (Typhoon 7600, GE Healthcare) if they werelabeled with fluorescence. The GST-HaloTag protein labeled wasvisualized by staining with Coomassie brilliant blue (CBB). The SDS-PAGEanalysis showed a fluorescent band (51 kDa) that corresponds to theTESRA-labeled HaloTag protein in nearly 40% of total labeling efficiency(cf, Lane 3 in FIG. 4). This efficiency was estimated by taking asalmost 100% the case where an azido-free HaloTag-Sepharose resin isfluorescence labeled with TESRA-HaloTag ligand (10) (cf, formula (26)).The fluorescence labeling efficiency of azido-free HaloTag-Sepharoseresin with TESRA-HaloTag ligand (10) was almost 100%, which wasconfirmed by MALDI-TOF-MS analysis as described below.

Modification of HaloTag Protein with TESRA-HaloTag Ligand (10)

First, the azido-free HaloTag-Sepharose resin was modified with theTESRA-HaloTag ligand (10). The modified HaloTag proteins were digestedwith a protease to elute from the Sepharose resin. The mass of theHaloTag proteins eluted was analyzed by MALDI-TOF-MS (cf, FIG. 5). InFIG. 5 showing the results of the mass spectrometry, line (1) (the peakof relative intensity is 35082 [a.u.]) and line (2) (the peak ofrelative intensity is 34355 [a.u.]) designate the molecular weight ofthe HaloTag protein modified with TERSRA-HaloTag ligand (10) and themolecular weight of unmodified HaloTag protein, respectively. As such,almost 100% peak shift was observed by the modification withTESRA-HaloTag ligand (10). The results indicate that the modificationefficiency with TESRA-HaloTag ligand (10) is almost 100%.

If any one of the azido-HaloTag ligand 8, diyne 3 and TESRA-PEO₃-azide(9) lacks, the GST-HaloTag protein was not fluorescence-labeled. Inother words, the fluorescence labeling is a reaction having particularlyhigh specificity that is almost free of non-specific reaction withrespect to proteins.

To confirm if the SPDC reaction proceeds as expected, the molecularweight of the azido-HaloTag protein labeled with TESRA by the SPDCreaction described above (EXAMPLE 21) was analyzed by MALDI-TOF-MS. Byexcising the junction region of GST and HaloTag proteins through limiteddigestion with a protease, each HaloTag protein was eluted from theGSH-Sepharose resin. The eluate was directly subjected to MALDI-TOF-MSanalysis. The results of this mass spectrometry are shown in FIG. 6. InFIG. 6, line (1) (the peak of relative intensity is 34355 [a.u.])designates the molecular weight of unreacted HaloTag protein and line(2) (the peak of relative intensity is 34357 [a.u.]) designates themolecular weight of azido-free HaloTag proteins obtained by reactingTESRA-PEO₃-azide (9) with diyne 3. Also, line (3) (the peak of relativeintensity is 34700 [a.u.]) designates the molecular weight of HaloTagprotein labeled with azido-HaloTag ligand 8 (azido-HaloTag protein) andline (4) (the peaks of relative intensity are 34699 and 35665 [a.u.])designates the molecular weights of azido-HaloTag proteins obtained byreacting azido-HaloTag protein with TESRA-PEO₃-azide (9) and diyne 3,respectively. Changes in molecular weight of these azido-HaloTagproteins by the SPDC reaction almost matched with the expected change inmolecular weight (35643 [a.u.]) by covalent bond of TESRA-PEO₃-azide (9)and diyne 3. It was therefore revealed that the SPDC reaction for theazido-HaloTag proteins proceeded smoothly as expected.

In the reaction described above, the reaction was allowed to proceed ina state where the azido-HaloTag protein (azido-HaloTag-Sepharose resin),diyne 3 and fluorescent azide molecule (TESRA-PEO₃-azide (9)) wereco-present in one test tube (simultaneous SPDC reaction). It is thusdifficult to artificially control the progress of the two clickreactions in the SPDC reaction. However, the inventors have found thatthe two click reactions of the SPDC reaction can be controlled. Aspecific procedure is given below. The azido-HaloTag-Sepharose resin wastreated with diyne 3. The unreacted diyne 3 was then quickly washed outby buffer. In this state it is expected that the monoyne intermediate inwhich the triple bond on one side of diyne 3 is conjugated with theazido-HaloTag-Sepharose would be present. Unexpectedly from thesimultaneous SPDC reaction described above, fluorescence labeling of theazido-HaloTag protein was achieved with an efficiency of approximately45% (cf., FIG. 4, Lane 7) even by sequential procedures of immediatelyadding TESRA-PEO₃-azide (9) in this state (sequential SPDC reaction).That is, it was demonstrated that the monoyne intermediate wherein onlyone side in diyne 3 was reacted could certainly be present.

Furthermore, in the simultaneous and sequential SPDC reactions describedabove, the signal for the homo-dimer of the azido-HaloTag protein washardly detected. These results indicate that the SPDC reaction betweenazido-proteins does not proceed at least under the conditions examined.This means that formation of the homo-dimer of the azido-protein, whichis a relatively large molecule, is almost certainly blocked under theSPDC reaction conditions for the proteins described above, indicatingthat efficient chemical modification can be achieved. Particularly inthe simultaneous SPDC reaction where the azido-HaloTag protein, diynecompound and fluorescent azide molecule are co-present, theazido-HaloTag protein and the fluorescent azide molecule can be added tothe diyne compound substantially simultaneously in one step, which makeschemical modification more efficient.

Synthesis of Azido-HaloTag Ligand 8

Under argon atmosphere, to a solution of tert-butylN-[2-{2-(6-chlorohexyloxy)ethoxy}ethyl]carbamate (11) (synthesized bythe method described in Non-Patent Document Y. Zhang, M.-k. So, A. M.Loening, H. Yao, S. S. Gambhir, J. Rao, Angew. Chem. Int. Ed., 2006,118, 4936-4940) (142 mg, 438 p.mol) in dichloromethane (3.5 mL) wasadded trifluoroacetic acid (0.5 mL) at 0° C. After stirring for 2 hoursat the same temperature of 0° C., the starting compound II completelydisappeared as judged from TLC study (R_(f)=0.41,dichloromethane/methanol=9/1). The reaction solution was thenconcentrated under reduced pressure using an evaporator to give crude2-[2-(6-chlorohexyloxy)ethoxy]ethylammonium trifluoroacetate (12) as acolorless oil. The product was used in the next step without furtherpurification.

Under argon atmosphere, to a solution of succinimido4-(azidomethyl)benzoate (13) (synthesized by the method described inNon-Patent Document A. Gopin, S. Ebner, B. Attali, D. Shabat,Bioconjugate Chem. 2006, 17, 1432-1440) (100 mg, 365 timol) indichloromethane (2 mL) were successively added triethylamine (153 μL,1.09 mmol) and a solution of the crude 12 prepared above at roomtemperature. After stirring for 18 hours at the same temperature, to themixture was added water (15 mL) and the product was extracted withdichloromethane (x3). The organic layer was washed with water (x1) anddried over anhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure using an evaporator and the residuewas purified by flash column chromatography (silica-gel 10 g,n-hexane/ethyl acetate=1/1) to give4-(azidomethyl)-N[2-{2-(6-chlorohexyloxy)ethoxy}ethyl]benzamide 8 (136mg, 355 μmol, 97.3% based on 13) as a yellow oil.

R_(f)=0.29 (hexane/ethyl acetate=1/1);

¹H NMR (300 MHz, CDCl₃) δ 1.28-1.50 (m, 4H), 1.51-1.66 (m, 2H),1.67-1.82 (m, 2H), 3.46 (t, 2H, J=6.7 Hz), 3.52 (t, 2H, J=6.7 Hz),3.57-3.62 (m, 2H), 3.64-3.71 (m, 6H), 4.40 (s, 2H), 6.75 (br s, 1H),7.36-7.42 (AA′BB′, 2H), 7.79-7.85 (AA′BB′, 2H);

¹³C NMR (75.5 MHz, CDCl₃) δ 25.3, 26.6, 29.3, 32.4, 39.6, 45.0, 54.1,69.6, 69.9, 70.1, 71.2, 127.5 (2C), 128.0 (2C), 134.4, 138.7, 166.8;

IR (KBr, cm⁻¹) 557, 650, 733, 754, 853, 908, 1018, 1115, 1200, 1252,1300, 1350, 1456, 1504, 1541, 1614, 1643, 2099, 2862, 2936, 3065, 3331;

HRMS (EI) m/z 382.1775 (M, C₁₈H₂₇ ³⁵ClN₄O₃ Calcd. 382.1772).

Synthesis of TESRA-PEO₃-azide (9)

Under argon atmosphere, to a solution of lissamine rhodamine B sulfonylchloride (15) (synthesized by the method described in Non-PatentDocument H. Yang, S. Vasudevan, C. O. Oriakhi, J. Shields, R. G. Carter,Synthesis 2008, 957-961) (600 mg, 1.04 mmol) in dichloromethane (30 mL)were successively added triethylamine (291 μL, 2.08 mmol) and11-azido-3,6,9-trioxaundecan-1-amine (14, commercial product) (90%) (275μL, 1.25 mmol) at 0° C. The mixture was allowed to warm to roomtemperature and stirred for 22 hours. The reaction solution wasconcentrated under reduced pressure using an evaporator. The residualsolid was placed on a Kiriyama funnel and then washed with ethylacetate. The collected solid was purified by flash column chromatography(silica-gel 50 g, dichloromethane/methanol=19/1) to giveTESRA-PEO₃-azide (9) (421 mg, 554 μmol, 53.4%) as a purple solid.

R_(f)=0.61 (dichloromethane/methanol=6/1);

¹H NMR (300 MHz, DMSO-d₆) δ 1.20 (t, 12H, J=7.1 Hz), 3.01 (br s, 2H),3.32-3.68 (m, 22H), 6.86-7.07 (m, 6H), 7.46 (d, 1H, J=7.7 Hz), 7.94 (d,1H, J=7.7 Hz), 8.06 (br s, 1H), 8.40 (s, 1H);

IR (KBr, cm⁻¹) 613, 683, 1024, 1074, 1134, 1182, 1197, 1248, 1281, 1341,1352, 1420, 1466, 1526, 1595, 1647;

UV (methanol) λmax (loge) 560.5 nm (5.16);

FL (methanol) λmax Em. 577 nm (Ex. 450 nm);

HRMS (ESI⁺) m/z 781.2649 ([M+Na]⁺, C₃₅H₄₆N₆NaO₉S₂ ⁺ Calcd. 781.2660).

Synthesis of TESRA-HaloTag Ligand (10)

Under argon atmosphere, to a solution of tert-butylN-[2-{2-(6-chlorohexyloxy)ethoxy}ethyl]carbamate (11) (synthesized bythe method described in Non-Patent Document Y. Zhang, M.-k. So, A. M.Loening, H. Yao, S. S. Gambhir, J. Rao, Angew. Chem. Int. Ed., 2006,118, 4936-4940) (84.2 mg, 260 μmol) in dichloromethane (2.5 mL) wasadded trifluoroacetic acid (0.5 mL) at 0° C. After stirring for 2 hoursat the same temperature of 0° C., the starting compound II completelydisappeared as judged from TLC study (R_(f)=0.41,dichloromethane/methanol=9/1). The reaction solution was thenconcentrated under reduced pressure using an evaporator to give crude2-[2-(6-chlorohexyloxy)ethoxy]ethylammonium trifluoroacetate (12) as acolorless oil. The crude product was used in the next reaction withoutfurther purification.

Under argon atmosphere, to a solution of lissamine rhodamine B sulfonylchloride (15) (100 mg, 173 μmol) in dichloromethane (2 mL) weresuccessively added triethylamine (72.4 pt, 519 μmol) and a solution ofthe crude product 12 obtained above in dichloromethane (1.5 mL) at roomtemperature. After stirring for 18 hours at the same temperature, thereaction solution was concentrated under reduced pressure using anevaporator. The residue was purified by flash column chromatography(silica-gel 10 g, dichloromethane/methanol=15/1) to give TESRA-HaloTagligand (10) (30.1 mg, 39.8 vimol, 22.8% based on 15) as a purple solid.

R_(f)=0.54 (dichloromethane/methanol=6/1);

¹H NMR (300 MHz, DMSO-d₆) δ 1.20 (t, 12H, J=7.0 Hz), 1.25-1.40 (m, 4H),1.42-1.52 (m, 2H), 1.62-1.73 (m, 2H), 3.01 (br s, 2H), 3.42-3.51 (m,8H), 3.55-3.70 (m, 10H), 6.90-7.10 (m, 6H), 7.45 (d, 1H, J=7.7 Hz), 7.94(d, 1H, J=7.7 Hz), 8.06 (br s, 1H), 8.40 (s, 1H);

IR (KBr, cm⁻¹) 579, 683, 1026, 1076, 1136, 1165, 1182, 1202, 1258, 1277,1350, 1396, 1420, 1466, 1483, 1524, 1597, 1645;

UV (methanol) λmax (logs) 561 nm (5.15);

FL (methanol) λmax Em. 577 nm (Ex. 450 nm);

HRMS (FAB⁺/NBA) m/z 764.2834 (M+H, C₃₇H₅₁ ³⁵ClN₃O₈S₂ Calcd. 764.2806).

Example 22 Fluorescence Labeling of Azido-Glycoconjugates on the Surfaceof Living Cells

To apply chemical modification to living cells by the SPDC reactionfurther in EXAMPLE 22, fluorescence labeling of azido-glycoconjugateslocalized on the cell surface was attempted. In EXAMPLE 22, HEK293 cellswere cultured for 2 days with a medium containing tetraacetylatedN-azidoacetyl-D-mannosamine (Ac₄ManNAz) and 10% fetal bovine serum in afinal concentration. Thereafter, the serum-containing culture medium wasexchanged with fresh medium, and diyne 3 was added thereto. The mixturewas allowed to stand at 37° C. for 20 minutes in the presence of 5%carbon dioxide. Immediately thereafter, the mixture was washed withfresh serum-containing culture medium to remove unreacted diyne 3. Thisprocedure was performed to prevent the side reaction that the azidecompound only is added to the cyclic diyne compound. Immediatelythereafter, TESRA-PEO₃-azide (9) (cf., formula (26) above) was added andthe mixture was allowed to stand at 37° C. for 20 minutes in thepresence of 5% carbon dioxide. To confirm the morphology of cells or thelocation of nuclei, the cells above were fixed and then further stainedwith Alexa Fluor 488-labeled phalloidin, which is a reagent forcytoskeleton staining, and a nuclear staining reagent TO-PRO-3. Thesefluorescence-labeled cells were observed on a laser-scanning confocalmicroscopy manufactured by Olympus Corporation, and visualized using anAdobe Photoshop CS2 software. As a result, a reliable fluorescencesignal was detected in the boundary between the cells where azidosugarsare incorporated into glycoconjugates on the cell surface (cf., thesuperimposed images in FIG. 7A, upper right corner). In contrast, in thecells where azidosugars are not incorporated into glycoconjugates on thecell surface (cf, buffer (−) in FIG. 7A, lower side), only a trace offluorescence signal was barely detected at almost a negligible level. Itis thus revealed that fluorescence labeling of the cell surface can beachieved only in the presence of azido-glycoconjugates and non-specificmodification to cells was hardly observed.

In the fluorescence labeling test system for the azido-glycoconjugatesin the HEK293 cells described above, the optimum results were obtainedwhen diyne 3 was used at concentrations of 40 to 100 μM. It was thusconfirmed that the labeling efficiency with diyne 3 was dependent onconcentration (cf., FIG. 7B). To determine the fluorescence labelingefficiency, intensities of the fluorescence images obtained weredigitalized using an Image-J software and represented graphically usingaveraged values. Standard error at each point is shown as well.

On the other hand, to optimize the reaction time of diyne 3, 40 μM ofdiyne 3 was added to the serum-containing culture medium of HEK293 cellswith azido-glycoconjugates, and the mixture was allowed to stand for 0to 48 minutes. Thereafter, fluorescence labeling was performed usingTESRA-PEO₃-azide (9) as described above. The fluorescence imagesobtained were quantified by the procedure described above andrepresented graphically. The results reveal that the efficiency becamemaximum in the reaction time for approximately 20 minutes but theefficiency decreased in the reaction time exceeding 20 minutes (cf.,FIG. 7C). This decrease in the labeling efficiency due to the passage oftime shows instability of the monoyne intermediate that only one side ofdiyne 3 has reacted with an azido group and is considered to be becausethe monoyne intermediate is degraded with the passage of time.

In order to study if not only the TESRA-PEO₃-azide (9) independentlysynthesized by the inventors but also low molecular azide compoundscommercially available can be used for the SPDC reaction, fluorescencelabeling tests were conducted on HEK293 cells withazido-glycoconjugates, using commercially available Alexa Fluor 488azide. As a result, fluorescence labeling could be efficiently achievedas well not only by the sequential procedures described above but alsoby the simultaneous procedures of adding diyne 3 and Alexa Fluor 488azide to the cell culture medium as shown in FIG. 8. FIG. 8 shows thesurface of HEK293 cells (azido-incorporated) labeled with Alexa Fluor488 azide and cell nuclei, and the surface of unlabeled cells and cellnuclei (azido-free), respectively. The foregoing results indicate thatthere is no big limitation to low molecular azide compounds used forchemical modification but diverse low molecular azide compounds may beused.

Furthermore, the inventors made comparative studies between themodification by the SPDC reaction and the modification by the singleclick reaction using the existing monoyne fluorescence derivatives. Theresults are shown in FIG. 9. Herein, fluorescence labeling was performedfor the azidosugar-incorporated HEK293 cells (azido-incorporated) andazidosugar-not incorporated HEK293 cells (azido-free), respectively, bythe SPDC reaction using diyne 3 and fluorescein-conjugated azide 18 andby the single click reaction using monoyne 22 (cf, reaction scheme 31)which is a fluorescence derivative of monoyne 2a. The SPDC reaction wascarried out in two types of reactions by simultaneous and sequentialprocedures. The respective reaction times are shown in the figure. As aresult of the comparative test, substantial difference in fluorescencelabeling efficiency between the SPDC reaction and the single clickreaction was hardly observed at the same reaction time. The resultsshowed a good match with the competition experiment demonstrated inEXAMPLE 19 and the results of the kinetic study shown in EXAMPLE 20 (cf,Paragraphs [0046] to [0048] described above and FIGS. 2 and 3). That is,it was demonstrated that the SPDC modification reaction using thefluorescein-conjugated azide 18 for diyne 3 and fluorescein-conjugatedmonoyne 22 described above to monoyne 2a proceeded at almost the samerate.

Synthesis of Fluorescein-Conjugated Azide 18

Under argon atmosphere, to a solution of 2-azidoethylamine (16) (˜50%,¹H NMR) (synthesized by the method described in Non-Patent Document: R.Srinivasan, L. P. Tan, H. Wu, P. Yang, K. A. Kalesh, S. Q. Yao, Org.Biomol. Chem. 2009, 7, 1821-1828) (200 mg, 1.16 mmol) in dichloromethane(5 mL) were successively added triethylamine (70.0 μL, 502 pimp andfluorescein isothiocyanate isomer I (FITC, 17, commercial product) (90%,HPLC) (100 mg, 231 μmol) at room temperature. After stirring for 21hours at the same temperature, the reaction solution was concentratedunder reduced pressure using an evaporator. The residue was purified byflash column chromatography (silica-gel 30 g,dichloromethane/methanol=9/1) to give1-(2-azidoethyl)-3-(5-fluoresceinyl)thiourea (18) (109 mg, 229 μmol,99.2%) as an orange solid.

TLC R_(f)=0.61 (dichloromethane/methanol=6/1);

¹H NMR (300 MHz, DMSO-d₆) δ 2.73 (t, 2H, J=5.8 Hz), 3.56 (t, 2H, J=5.8Hz), 3.65-3.80 (br, 2H), 6.54 (dd, 2H, J=2.2, 8.6 Hz), 6.61 (d, 2H,J=8.6 Hz), 6.65 (d, 2H, J=2.2 Hz), 7.18 (d, 1H, J=8.4 Hz), 7.72 (dd, 1H,J=1.7, 8.4 Hz), 8.21 (d, 1H, J=1.7 Hz), 8.25-8.40 (br, 1H), 9.80-10.60(br, 1H);

IR (KBr, cm⁻¹) 459, 476, 577, 600, 667, 812, 851, 914, 1111, 1173, 1207,1296, 1389, 1458, 1570, 2108, 2930;

UV (methanol) λmax (logs) 456 nm (4.07), 482 nm (4.09);

FL (methanol) λmax Em. 516.5 nm (Ex. 450 nm);

HRMS (ESI⁺) m/z 476.1024 ([M+H]⁺, C₂₃H₁₈N₅O₅S⁺ required 476.1023).

Synthesis of Fluorescein-Conjugated Monoyne 22

Under argon atmosphere, to a solution of ethylenediamine (19) (320 μL,479 μmol) in dichloromethane (5 mL) were successively addedtriethylamine (330 μL, 237 μmol) and11,12-didehydro-5,6-dihydrodibenzo[a,e]cycloocten-5-yl 4-nitrophenylcarbonate (20) (synthesized by the method described in Non-PatentDocument X. Ning, J. Guo. M. A. Wolfert, G.-J. Boons, Angew. Chem. Int.Ed. 2008, 47, 2253-2255) (60.3 mg, 156 μmol) at room temperature. Afterstirring for 2 hours at the same temperature, the starting compound 20completely disappeared as judged from the TLC study. To the reactionsolution was added dichloromethane (70 mL) and the mixture was washedwith saturated aqueous sodium hydrogen carbonate solution (20 mL x7),dried over anhydrous sodium sulfate and filtered. The filtrate wasconcentrated under reduced pressure using an evaporator to give crude11,12-didehydro-5,6-dihydrodibenzo[a,e]cycloocten-5-ylN-(2-azidoethyl)carbamate (21) (51.2 mg) as a pale yellow oil, which wasused in the next step without further purification.

Under argon atmosphere, to a solution of the crude 21 obtain as above inDMF (3 mL) were successively added triethylamine (29.0 μL, 208 μmol) andfluorescein isothiocyanate isomer I (FITC, 17, commercial product) (90%,HPLC) (45.0 mg, 104 μmol) at room temperature. After stirring for 17hours at the same temperature, the reaction solution was concentratedunder reduced pressure. The residue was purified by flash columnchromatography (silica-gel 8 g, dichloromethane/methanol=12/1) to give1-[2-{(11,12-didehydro-5,6-dihydrodibenzo[a,e]cycloocten-5-yl)oxycarbonylamino}ethyl]-3-(5-fluoresceinyl)thiourea(22) (59.0 mg, 84.8 μmol, 80.7% based on 17) as a yellow solid.

TLC R_(f)=0.52 (dichloromethane/methanol=6/1);

¹H NMR (300 Mz, CD₃OD) δ 2.73 (dd, 1H, J=3.7, 14.9 Hz), 3.20 (dd, 1H,J=2.4, 14.9 Hz), 3.70-3.80 (m, 2H), 5.28 (s, 1H), 6.41 (dd, 2H, J=2.3,9.2 Hz), 6.59 (dd, 2H, J=2.3, 9.2 Hz), 6.70 (d, 1H, J=2.3 Hz), 6.71 (d,1H, J=2.3 Hz), 6.97-7.56 (m, 12H), 7.83 (s, 1H);

IR (KBr, cm⁻¹) 488, 546, 565, 578, 760, 851, 914, 993, 1022, 1076, 1113,1179, 1207, 1258, 1317, 1449, 1506, 1595, 1701, 2938, 3063, 3287;

UV (methanol) λmax (loge) 455 nm (3.95), 481 nm (3.95);

FL (methanol) λmax Em. 515 nm (Ex. 450 nm);

HRMS (ESI⁺) m/z 696.1785 ([M+H]⁺, C₄₀H₃₀N₃O₇S⁺ required 696.1799).

Finally, cytotoxicity assay of diyne 3 was performed in living cells.HEK293 cells were incubated and diyne 3 was added to the cells in aconcentration of 0 to 80 μM, followed by incubation overnight.Morphological images of the cells after incubation, which were takenunder a phase-contrast microscope and visualized with Adobe PhotoshopCS2, are shown in FIG. 10. The results indicate that diyne 3 did notshow cytotoxicity and is applicable to chemical modification of livingcells as well.

As demonstrated above, it was revealed that diyne 3 is useful as asubstrate in the modification of azido-biomolecules with the functionalazide compound having a fluorescent functional group, etc. as a probe,taking advantage of the SPDC reaction. In particular, diyne 3 (or cyclicdiyne compounds having similar structures) and a variety of functionalazido-compounds are readily available or can be easily synthesized.Thus, various functional biomolecules can be prepared by applying theSPDC reaction. Furthermore, two azide compounds can be added and ligatedin a single step and by utilizing the SPDC reaction which rapidlyproceeds even under catalyst-free, mild conditions, functionalbiomolecules can be prepared easily and efficiently even in livingcells, etc. while maintaining their functions.

1-5. (canceled)
 6. A method for producing a cyclic compound, whichcomprises adding and ligating an azide compound having an azido group toeach of the two carbon-carbon triple bond sites in a cyclic diynecompound by a double click reaction to produce a cyclic compoundcomprising two triazole rings.
 7. A method for modifying a biomolecule,which comprises adding and ligating the azido group of an azide compoundas a probe and the azido group incorporated into the biomolecule to eachof the two carbon-carbon triple bond sites in a cyclic diyne compound bya double click reaction to produce a cyclic compound.
 8. The modifyingmethod according to claim 7, wherein the cyclic diyne compound has an8-membered cyclic skeleton comprising two carbon-carbon triple bondsites.
 9. The modifying method according to claim 8, wherein the cyclicdiyne compound further has a benzene ring and/or heteroaromatic ringsharing the carbon-carbon double bond sites with the cyclic skeleton.10. The modifying method according to claim 7, wherein the cyclic diynecompound is represented by formula (3) below:

(in formula (3), each R independently represents hydrogen or ahydrocarbon group, and each n independently represents an integer of 0to 4).
 11. The modifying method according to claim 7, wherein theaddition reaction of the azide compound and the biomolecule proceedswithout using any catalyst.
 12. The modifying method according to claim7, wherein the double click reaction is performed in the co-presence ofthe azide compound and the biomolecule.
 13. The modifying methodaccording to claim 7, wherein the biomolecule is added to the cyclicdiyne compound, the unreacted cyclic diyne compound is removed and theazide compound is added to the biomolecule-added cyclic diyne compoundonly.